Methods and compositions for modulating the activity of the interleukin-35 receptor complex

ABSTRACT

The receptor for Interleukin 35 (IL-35) is provided. The Interleukin 35 Receptor (IL-35R) comprises a heterodimeric complex of the Interluekin12Rβ2 receptor and the gp130 receptor. Various compositions comprising the IL-35R complex, along with polynucleotides encoding the same and kits and methods for the detection of the same the same are provided. Methods of modulating the activity of IL-35R or modulating effector T cell functions are also provided. Such methods employ various IL-35R antagonists and agonists that modulate the activity of the IL-35R complex and, in some embodiments, modulate effector T cell function. Further provided are methods for screening for IL-35R binding agents and for IL-35R modulating agents. Various methods of treatment are further provided.

FIELD OF THE INVENTION

The present invention relates to methods for regulating T cell functionin a subject, particularly effector T cell activity.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 397057SEQLIST.txt, a creation date of Nov. 16, 2010, and a sizeof 61 KB. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

The immune system provides the human body with a means to recognize anddefend itself against microorganisms, viruses, and substances recognizedas foreign and potentially harmful. Classical immune responses areinitiated when antigen-presenting cells present an antigen to CD4+ Thelper (Th) lymphocytes resulting in T cell activation, proliferation,and differentiation of effector T lymphocytes. Following exposure toantigens, such as that which results from infection or the grafting offoreign tissue, naïve T cells differentiate into Th1 and Th2 cells withdiffering functions. Th1 cells produce interferon gamma (IFN-γ) andinterleukin 2 (IL-2) (both associated with cell-mediated immuneresponses). Th1 cells play a role in immune responses commonly involvedin the rejection of foreign tissue grafts as well as many autoimmunediseases. Th2 cells produce cytokines such as interleukin-4 (IL-4), andare associated with antibody-mediated immune responses such as thosecommonly involved in allergies and allergic inflammatory responses suchas allergic rhinitis and asthma. Th2 cells may also contribute to therejection of foreign grafts. In numerous situations, this immuneresponse is desirable, for example, in defending the body againstbacterial or viral infection, inhibiting the proliferation of cancerouscells and the like. However, in other situations, such effector T cellsare undesirable, e.g., in a graft recipient.

Whether the immune system is activated by or tolerized to an antigendepends upon the balance between T effector cell activation and Tregulatory cell activation. T regulatory cells are responsible for theinduction and maintenance of immunological tolerance. Regulatory T cellsactively suppress the proliferation and cytokine production of Th1, Th2,or naïve cells which have been stimulated in culture with an activatingsignal (e.g., antigen and antigen presenting cells or with a signal thatmimics antigen in the context of MHC, e.g., anti-CD3 antibody, plusanti-CD28 antibody).

Undesirable immune responses have generally been treated withimmunosuppressive drugs, which inhibit the entire immune system, i.e.,both desired and undesired immune responses. General immunosuppressantsmust be administered frequently, for prolonged periods of time, and havenumerous harmful side effects. Withdrawal of these drugs generallyresults in relapse of disease. Thus, there is a need for agents thatpreferentially modulate either the effector or the regulatory arm of theimmune system.

SUMMARY OF THE INVENTION

The receptor for Interleukin 35 (IL-35) is provided. The Interleukin-35Receptor (IL-35R) comprises a heterodimeric complex of theInterleukin-12Rβ2 receptor (Il12rb2) and the gp130 receptor (also knownas Interleukin-6 signal transducer, Il6st). Various compositionscomprising the IL-35R complex, along with polynucleotides encoding thesame and kits and methods for the detection of the same are provided.

Methods of modulating the activity of IL-35R or modulating effector Tcell functions are provided. Such methods employ various IL-35Rantagonists and agonists that modulate the activity of the IL-35Rcomplex and, in some embodiments, modulate effector T cell function.Further provided are methods for screening for IL-35R binding agents andfor IL-35R modulating agents. Various methods of treatment are furtherprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that T-cells that lack both the Interluekin12Rβ2receptor (IL12Rβ2) and the gp130 receptor are completely resistant tosuppression mediated by IL-35 or iTr35, an induced regulatory T cellpopulation that suppresses via IL-35.

FIG. 2 demonstrates that IL35R deficient T_(conv) are resistant to IL-35mediated suppression in vivo. Homeostatic expansion was monitored byi.v. injection of Thy1.2⁺ T_(conv) cells from wild-type (C57BL/6),gp130^(ΔT) (gp130 deficient in T cells), Il12rb2^(−/−) or IL35RAT(gp130^(ΔT)/Il12rb2^(−/−)) mice alone or with Thy1.1⁺ iT_(R)35 cells (asregulatory cells) into Rag1^(−/−) mice. Seven days after transfer,splenic T cell numbers were determined by flow cytometry. (B) Rag1^(−/−)mice received CD4⁺ and CD8⁺ T cells from wild-type (C57BL/6),gp130^(ΔT), Il12rb2^(−/−) or IL35R^(ΔT) mice alone or with iT_(R) ³⁵cells via the tail vein on day −1 of the experiment. On day 0, all wereinjected with 120,000 B16 cells i.d. in the right flank. Tumor diameterwas measured daily for 14 days and is reported as mm³. Data representthe mean±SEM of 5-12 mice per group.

FIG. 3 demonstrates that IL35 is a target of the IL35 signaling pathway.(A) T_(conv) purified by FACS from wild-type (C57BL/6), gp130^(ΔT)(gp130 deficient in T cells), Il12rb2^(−/−) or IL35R^(ΔT) (gp130^(ΔT)Il12rb2^(−/−)) mice were activated with anti-CD3-+anti-CD28-coated latexbeads for 18 hours in the presence of IL-35. RNA was extracted, cDNAgenerated and qPCR performed. Relative Ebi3 (left panel) and Il12a(right panel) mRNA expression. (B) T_(conv) from wild-type (C57BL/6),gp130^(ΔT), Il12rb2^(−/−) or IL35R^(ΔT) mice were activated in thepresence of IL-35 or control protein at 25% of total culture volume, for72 hours to generate iT_(R) ³⁵ or iT_(R)con cells, respectively. Cellswere re-purified and mixed at indicated ratios (T_(conv): suppressor)and proliferation was determined by [³H]-thymidine incorporation. Countsper minute of T_(conv) cells activated alone were 29,000-48,000 (b).Data represent the mean±SEM of 3-5 independent experiments.

DETAILED DESCRIPTION OF THE INVENTION I. Compositions

The receptor for Interleukin 35 (IL-35) is provided. As demonstratedherein, the Interleukin 35 Receptor (IL-35R) comprises a heterodimericcomplex of the Interluekin12Rβ2 receptor (IL12Rβ2) and the gp130receptor.

As used herein, the Interleukin 35 receptor (IL-35R) refers to anyintramolecular complex or single molecule comprising at least one gp130polypeptide component or biologically active variant or fragment thereofand at least one IL12Rβ2 polypeptide component or biologically activevariant or fragment thereof. Typically, in vivo, gp130 and IL12Rβ2associate via a non-covalent association. For purposes of the presentinvention, the IL12Rβ2 and gp130 components may be associated with oneanother either covalently or non-covalently. In some examples, gp130 andIL12Rβ2 can be co-expressed as a fusion protein.

Biologically active fragments and variants of the IL-35R complex arealso provided. Such IL-35R complexes comprise an active variant orfragment of gp130 and/or an active variant or fragment of IL12Rβ2 andwill retain at least one activity of the IL-35R complex.

The phrase “biological activity of IL-35R” refers to one or more of thebiological activities of IL-35R, including but not limited to, (1)interacting with its ligand, IL-35; (2) activating any of the STATpathways including the STAT1 and/or STAT4 pathways; (3) IL-35 dependentsuppression of effector T-cell function, including for example,suppression of proliferation, cytokine secretion and/or differentiation;and/or (4) autocrine induction of IL-35 expression by IL-35. Such assayscan be found, for example, in Collison et al. (2007) Nature 450:566-569;Collison et al. (2010) Nature Immunology 11: 1093-1101.

As discussed above, the IL-35R complex interacts with the ligand, IL-35.As used herein, “Interleukin 35” or “IL-35” refers to any intramolecularcomplex or single molecule comprising at least one EBI3 polypeptidecomponent and at least one p35 polypeptide component. See, for example,International Patent Application No. PCT/US2007/079310, filed Sep. 24,2007, herein incorporated by reference in its entirety. EBI3 and p35 areknown in the art. The human EBI3 gene encodes a protein of about 33 kDa.The protein shares about 27% sequence identity to the p40 subunit ofhuman IL12. Nucleic acid and amino acid sequences for EBI3 are known.See, for example, SEQ ID NOs:1 and 2 of WO97/13859 (human) and GenBankAccession Numbers NM015766 and BC046112 (mouse). Nucleic acid and aminoacid sequences for p35 are also known in the art and include SEQ IDNOs:3 and 4 of WO97/13859 (human) and GenBank Accession NumbersNM_(—)000882 and M86672 (mouse). The term IL-35 encompasses naturallyoccurring variants of IL-35, e.g., splice variants, allelic variants,and other isoforms. The term also encompasses fragments or variants of anative IL-35 such that the active variants and fragment continue to bindand allow for the activation of IL-35R.

It is recognized that an IL-35R complex can be encoded on a singlepolynucleotide. For example, in one embodiment, a polynucleotidecomprising a nucleotide sequence encoding an Interleukin 35 receptor(IL-35R) complex is provided and comprises a first sequence encoding thegp130 polypeptide or an active fragment or variant thereof; and a secondsequence encoding the IL12Rβ2 polypeptide or an active fragment orvariant thereof, wherein said encoded polypeptides form a biologicallyactive IL-35R complex. In another embodiment, the IL-35R complex isencoded on distinct polynucleotides. Thus, a mixture of recombinantexpression constructs encoding the various components of the IL-35Rcomplex are further provided.

i. IL12Rβ2 Polynucleotides and Polypeptides

The polypeptides that interact to form the IL-35R complex are known inthe art. As used herein, the terms “Interleukin12Rβ2 receptor”,“IL12Rβ2” or “IL-12R-beta2” can be used interchangeably and refer to afamily of cell surface receptors which can homodimerize orheterodimerize and display interleukin receptor activity and have nowbeen shown herein to be a subunit of the IL-35R complex. Non-limitingexamples of IL12Rβ2 polypeptides comprise the human IL12Rβ2polynucleotide as set forth in SEQ ID NO:4, 5, and 6 (from GenBankAccession No. NM_(—)001559.2) and can be found in GenBank Accession No.P40189.

IL12Rβ2 polypeptides comprise a variety of conserved structural motifs.For ease of reference, such motifs will be discussed as they relate tothe human IL12Rβ2 isoform 1 which is set forth in SEQ ID NO:6. IL12Rβ2polypeptides comprise an extracellular domain (from about amino acids24-522 of SEQ ID NO:6); a transmembrane domain (from about amino acids623-643 of SEQ ID NO:3), and an intercellular domain (from about aminoacids 644-862 of SEQ ID NO:6). Additional conserved domains and motifsthat have been characterized in the IL12Rβ2 polypeptides include asignal peptide (from about amino acids 1-23 of SEQ ID NO:6), aFibronectin type III I domain (from about amino acids 124-218 of SEQ IDNO:6), a Fibronectin type III 2 domain (from about amino acids 224-316of SEQ ID NO:6), a Fibronectin type III 3 domain (from about amino acids317-415 of SEQ ID NO:6), a Fibronectin type III 4 domain (from aboutamino acids 420-517 of SEQ ID NO:6), a Fibronectin type III 5 domain(from about amino acids 521-617 of SEQ ID NO:6), a motif involved withSTAT4 binding (from about amino acids 796-801 of SEQ ID NO:6), a WSXWSmotif (from about amino acids 305-309 of SEQ ID NO:6) which appears tobe involved in proper protein folding and thereby efficientintracellular transport and cell-surface receptor binding, and a Box 1motif (from about amino acids 662-670 of SEQ ID NO:6) which is involvedin JAK interaction and/or activation. Glycosylation can occur at aminoacid positions 48, 129, 166, 195, 271, 347, 376, and 480 of SEQ ID NO:6.

It is recognized that biologically active variants and fragments of theIL12Rβ2 polypeptide can be employed in the various methods andcompositions of the invention. Such active variants and fragments willcontain an IL-35R receptor activity when complexed with the gp130partner. Variants of IL12Rβ2 are known including, but not limited to, analternative sequence from aa 650-659 of SEQ ID NO:6 replacing VFVLLAALRPwith RRHSCPWTGS; an alternative sequence from aa 660-862 of SEQ ID NO:6is missing; the Rat aa 149 of SEQ ID NO:6 is replaced with Q; the I ataa 185 of SEQ ID NO:6 is replaced with V; the T at aa 201 of SEQ ID NO:6is replaced with I; the R at aa 313 of SEQ ID NO:6 is replaced with G;the G at aa 420 of SEQ ID NO:6 is replaced with R; the Q at aa 426 ofSEQ ID NO:6 is replaced with H; the G at aa 465 of SEQ ID NO:6 isreplaced with D; the A at aa 625 of SEQ ID NO:6 is replaced with V; theH at aa 720 of SEQ ID NO:6 is replaced with R; the L at aa 808 of SEQ IDNO:6 is replaced with R; the Y at aa 678 of SEQ ID NO:6 is replaced withF; or the Y at aa 767 of SEQ ID NO:6 is replaced with F.

In specific embodiments, fragments of IL12Rβ2 are employed whichcomprise the extracellular domain of the IL12Rβ2 polypeptide or abiologically active fragment or variant of the extracellular domain ofIL12Rβ2. Such biologically active variants and fragments of the IL12Rβ2extracellular domain will retain the ability to complex with the gp130binding partner's extracellular domain and upon complex formation, thegp130/IL12Rβ2 complex will interact with the IL-35 ligand. Methods toassay for such binding are known.

Thus, in one embodiment, the IL12Rβ2 polypeptide comprises the aminoacid sequence as shown in SEQ ID NO:6 or a biologically active variantor fragment thereof. Further provided are polynucleotides comprising thenucleotide sequence encoding a IL12Rβ2 polypeptide including thenucleotide sequence set forth in SEQ ID NO:4 or 5 or a biologicallyactive variant or fragment thereof.

ii. Gp130 Polynucleotides and Polypeptides

As used herein, the terms “gp130”, “Interleukin-6-receptor subunitbeta”, “IL-6R-beta”, “Interleukin-6-signal transducer”, “membraneglycoprotein 130”, “CDW130”,

“Oncostatin-M receptor alpha subunit”, “CD_antigen=CD130” or IL6ST” canbe used interchangeably and refer to a family of cell surface receptorswhich can homodimerize or heterodimerize and display interleukinreceptor activity and have now been shown herein to be a subunit of theIL-35R complex. Non-limiting examples of gp130 polypeptides comprisingthe human gp130 polynucleotide and polypeptide are set forth in SEQ IDNOs: 1, 2, and 3 (GenBank Accession No. NP_(—)002184.2) or also inGenBank Accession No. P40189.

The gp130 polypeptide comprises a variety of conserved structural motifsand belongs to the type I cytokine receptor family. For ease ofreference, such motifs will be discussed as they relate to the humangp130 isoform 1 which is set forth in SEQ ID NO:3. gp130 polypeptidescomprise an extracellular domain (from about amino acids 23-619 of SEQID NO:3); a transmembrane domain (from about amino acids 260-641 of SEQID NO:3), and an intercellular domain (from about amino acids 642-918 ofSEQ ID NO:3). Additional conserved domains and motifs have beencharacterized in the gp130 polypeptides include a signal peptide (fromabout amino acids 1-22 of SEQ ID NO:3), an IG-like C2-type domain (fromabout amino acids 26-120 of SEQ ID NO:3), a Fibronectin type III Idomain (from about amino acids 125-216 of SEQ ID NO:3), a Fibronectintype III 2 domain (from about amino acids 222-321 of SEQ ID NO:3), aFibronectin type III 3 domain (from about amino acids 326-418 of SEQ IDNO:3), a Fibronectin type III 4 domain (from about amino acids 423-514of SEQ ID NO:3), a Fibronectin type III 5 domain (from about amino acids518-610 of SEQ ID NO:3), a WSXWS motif (from about amino acids 310-314of SEQ ID NO:3) which appears to be involved in proper protein foldingand thereby efficient intracellular transport and cell-surface receptorbinding, a Box 1 motif (from about amino acids 651-659 of SEQ ID NO:3)which is involved in JAK interaction and/or activation, and acompositional bias that is Ser-rich (from about amino acids 725-755 ofSEQ ID NO:3). Glycosylation can occur at amino acid positions 43, 83,131, 157, 227, 379, 383, 553, 564 of SEQ ID NO:3. Modified phosphoserineresidues can occur at amino acids 667, 782, 820, and 829 of SEQ ID NO:3.Disulfide bonds can occur between amino acids 28 and 54, 48 and 103, 134and 144, 172 and 182, and 458 and 466 of SEQ ID NO:3.

It is recognized that biologically active variants and fragments of thegp130 polypeptide can be employed in the various methods andcompositions of the invention. Such active variants and fragments willcontinue to retain an IL-35R activity when complexed with the IL12Rβ2partner. Variants and fragments of gp130 polypeptides andpolynucleotides are known including, but not limited to, an alternativesequence from aa 325-329 of SEQ ID NO:3 replacing RPSKA with NIASF; analternative sequence from aa 330-918 of SEQ ID NO:3 is missing; the T ataa 415 of SEQ ID NO:3 is replaced with I; or the S at aa 782 of SEQ IDNO:3 is replaced with A.

In specific embodiments, fragments of the gp130 are employed whichcomprise the extracellular domain of the gp130 polypeptide or abiologically active fragment or variant of the extracellular domain ofgp130. Such biologically active variants and fragments of theextracellular domain of gp130 will retain the ability to complex withthe IL12Rβ2 binding partner's extracellular domain and upon complexformation, the gp130/IL12Rβ2 complex can interact with the IL-35 ligand.Methods to assay for such binding are known.

Thus, in one embodiment, the gp130 polypeptide comprises the amino acidsequence as shown in SEQ ID NO:3 or a biologically active variant orfragment thereof. Further provided are polynucleotides comprising thenucleotide sequence encoding a gp130 polypeptide including thenucleotide sequence set forth in SEQ ID NO:1 or 2 or a biologicallyactive variant or fragment thereof

iii. Variants and Fragments

Fragments and variants of the polynucleotides encoding the gp130 andIL12Rβ2 polypeptides can be employed in the various methods andcompositions of the invention. By “fragment” is intended a portion ofthe polynucleotide and hence the protein encoded thereby or a portion ofthe polypeptide. Fragments of a polynucleotide may encode proteinfragments that retain the biological activity of the native protein andhence have IL-35R activity when complexed with the appropriate bindingpartner. Thus, fragments of a polynucleotide may range from at leastabout 20 nucleotides, about 50 nucleotides, about 100 nucleotides, about150, about 200, about 250, about 300, about 350, about 400, about 450,about 500, about 550, about 600 and up to the full-length polynucleotideencoding the gp130 or IL12Rβ2 polypeptide.

A fragment of a polynucleotide that encodes a biologically activeportion of a gp130 or IL12Rβ2 polypeptide will encode at least 15, 25,30, 50, 100, 150, 200, or 250 contiguous amino acids, or up to the totalnumber of amino acids present in a full-length gp130 and IL12Rβ2polypeptide.

A biologically active portion of a gp130 or IL12Rβ2 polypeptide can beprepared by isolating a portion of one of the polynucleotides encodingthe portion of the gp130 or IL12Rβ2 polypeptide and expressing theencoded portion of the polypeptide (e.g., by recombinant expression invitro), and assessing the activity of the portion of the gp130 orIL12Rβ2 polypeptide. Polynucleotides that encode fragments of a gp130 orIL12Rβ2 polypeptide can comprise nucleotide sequence comprising at least16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 nucleotides, orup to the number of nucleotides present in a full-length gp130 andIL12Rβ2 nucleotide sequence disclosed herein.

“Variant” sequences have a high degree of sequence similarity. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the gp130 or IL12Rβ2 polypeptides. Variants such asthese can be identified with the use of well-known molecular biologytechniques, as, for example, polymerase chain reaction (PCR) andhybridization techniques. Variant polynucleotides also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode agp130 or a IL12Rβ2 polypeptide. Generally, variants of a particularpolynucleotide will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to that particular polynucleotide as determinedby sequence alignment programs and parameters described elsewhereherein.

Variants of a particular polynucleotide can also be evaluated bycomparison of the percent sequence identity between the polypeptideencoded by a variant polynucleotide and the polypeptide encoded by thereference polynucleotide. Thus, for example, isolated polynucleotidesthat encode a polypeptide with a given percent sequence identity to thegp130 and IL12Rβ2 polypeptides set forth herein. Percent sequenceidentity between any two polypeptides can be calculated using sequencealignment programs and parameters described. Where any given pair ofpolynucleotides is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins arebiologically active, that is they continue to possess the desiredbiological activity of the native protein, that is, IL-35R activity.Such variants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a gp130 or IL12Rβ2polypeptides will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a protein may differfrom that protein by as few as 1-15 amino acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue.

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the gp130 or IL12Rβ2 proteins can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferable.

Thus, the polynucleotides used in the invention can include thenaturally occurring sequences, the “native” sequences, as well as mutantforms. Likewise, the proteins used in the methods of the inventionencompass naturally occurring proteins as well as variations andmodified forms thereof. Such variants will continue to possess theability to implement a recombination event. Generally, the mutationsmade in the polynucleotide encoding the variant polypeptide should notplace the sequence out of reading frame, and/or create complementaryregions that could produce secondary mRNA structure. See, EP PatentApplication Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different gp130 orIL12Rβ2 coding sequences can be manipulated to create a new gp130 orIL12Rβ2 polypeptides possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

iv. IL-35R Binding and/or Modulating Agents

1. Modulating Agents

As used herein, the term “modulating” includes “inducing”, “inhibiting”,“potentiating”, “elevating”, “increasing”, “decreasing” or the like.Each of these terms denote a quantitative difference between two statesand in particular, refer to at least a statistically significantdifference between the two states.

The term “IL-35R agonist” refers to an agent which potentiates, inducesor otherwise enhances one or more of the biological properties of theIL-35R complex. The activity increases by a statistically significantamount including, for example, an increase of at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 95% or 100% of the activity of the IL-35R complex compared to anappropriate control.

The term “IL-35R antagonist” refers to an agent which reduces, inhibits,or otherwise diminishes one or more of the biological activities of theIL-35R complex. Antagonism using the IL-35R antagonist does notnecessarily indicate a total elimination of the IL-35R activity.Instead, the activity could decrease by a statistically significantamount including, for example, a decrease of at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 95% or 100% of the activity of the IL-35R complex compared to anappropriate control.

By “specific modulating agent” is intended an agent that modulates theactivity of a defined target. Thus, an IL-35R specific modulating agentmodulates the biological activity of IL-35R by a statically significantamount (i.e., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100% or greater) and the agent does not modulate the biological activityof any monomeric subunits, homodimeric complexes or non-IL-35Rheterodimeric complexes which comprise either IL12Rβ2 or gp130 by astatistically significant amount (i.e., the activity is modulated byless than 5%, 4%, 3%, 2% or 1%). One of skill will be aware of theproper controls that are needed to carry out such a determination. AnIL-35R specific modulating agent may or may not be an IL-35R specificbinding agent.

In one non-limiting embodiment, the IL-35R modulating agent comprises asoluble IL-35R complex. Such a soluble complex is an IL-35R. As usedherein, a “soluble IL-35R complex” comprises an IL-35R polypeptide thatis incapable of anchoring itself in a membrane. Such soluble IL-35Rpolypeptides include, for example, a complex of gp130 and/or IL12Rβ2polypeptides that lack a sufficient portion of their membrane spanningdomain to anchor the IL-35R to the membrane or such polypeptides aremodified such that the membrane spanning domain is non-functional. Forexample, a soluble fragment of a gp130 polypeptide comprises theextracellular domain of gp130, including a fragment of the extracellulardomain that is at least 20, 30, 40, 50, 60, 70, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 590 or greater consecutive amino acids ofgp130. A soluble fragment of an IL12Rβ2 polypeptide comprises theextracellular domain of IL12Rβ2, including a fragment of theextracellular domain that is at least 20, 30, 40, 50, 60, 70, 90, 100,150, 200, 250, 300, 350, 400, 450, 495 or greater consecutive aminoacids of IL12Rβ2. In specific embodiments, the soluble IL-35R complexbinds IL-35. In other embodiments, the extracellular domains of gp130and IL12Rβ2 that are present in the soluble form of the IL-35R complexshare at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% to the amino acid sequence or the polynucleotide sequencesas set forth in SEQ ID NO: 1, 2, 3, 4, 5 or 6. The soluble IL-35Rcomplex find further use in stabilizing IL-35 (increasing half-life) oracting to concentrate IL-35.

A soluble IL-35R complex can additionally include a second moiety. Thesecond moiety can be any chemical compound. In specific embodiments, thesecond moiety adds in the detection of the soluble complex or promotesthe overall solubility of the complex. Such moieties include, but arenot limited to, an immunoglobulin chain, a GST, Lex-A or MBP polypeptidesequence. For example, a fusion protein can includes at least a fragmentof an IL-35R complex, which is capable of binding IL-35, wherein theIL-35R complex comprises a soluble fragment of a gp130 (e.g., a fragmentof gp130 comprising the extracellular domain of gp130) and a solublefragment of IL12Rβ2 (e.g., a fragment of IL12Rβ2 comprising theextracellular domain of IL12Rβ2) wherein at least one of the gp130fragment, the IL12Rβ2 fragment, or both are fused to a second moiety. Inspecific embodiments, the second moiety comprises an immunoglobulinchain, an Fc fragment (CH₂, hinge, CH₃ constant region domains), a heavychain constant region domain of the various isotypes, including: IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE).

A soluble form of the IL-35R complex can be generated using variousprotein motifs that assist in complex formation. One such motifcomprises a leucine zipper motif. Leucine zipper domains are peptidesthat promote oligomerization of the polypeptides in which they arefound. Leucine zippers were originally identified in several DNA-bindingpolypeptides (Landschulz et al. (1988) Science 240:1759), and have sincebeen found in a variety of different polypeptides. Among the knownleucine zippers are naturally occurring peptides and derivatives thereofthat dimerize or trimerize. The zipper domain (also referred to hereinas an oligomerizing, or oligomer-forming, domain) comprises a repetitiveheptad repeat, often with four or five leucine residues interspersedwith other amino acids. Use of leucine zippers and preparation ofoligomers using leucine zippers are well known in the art. Any othermethod which assists in the stabilization of the soluble complex can beemployed.

It is recognized that a soluble IL-35R complex can be encoded on asingle polynucleotide. For example, in one embodiment, a polynucleotidecomprising a nucleotide sequence encoding a soluble Interleukin 35receptor (IL-35R) complex is provides and comprise a first sequenceencoding the extracellular domain of gp130, an active fragment orvariant thereof; and a second sequence encoding the extracellular domainof IL12Rβ2, active fragment or variant thereof, the encoded solublepolypeptide complex bind IL-35. In another embodiment, the solubleIL-35R complex is encoded on distinct polynucleotides.

In another embodiment, a “specific modulating agent” can comprise anagent, such as an antibody, which modulates the ability of the IL-35R tobe activated by IL-35 but permits the IL-35R complex to be activated byother non-IL-35 ligands. Such agents can be IL-35R binding agents, agp130 binding agent, or an IL12Rβ2 binding agent.

2. IL-35R Binding Agents As used herein, an “IL-35R binding agent”refers to any compound that directly interacts with or binds to theIL-35R complex. By “specific binding agent” is intended an agent thatbinds substantially only to a defined target. Thus, an IL-35R specificbinding agent interacts directly with IL-35R and binds substantiallyonly to epitopes which are formed upon the interaction of IL12Rβ2 andgp130 to form the biologically active IL-35R. Thus, an IL-35R specificbinding agent will not substantially interact with monomeric proteinsubunits comprising IL12Rβ2 or gp130 and the agent will notsubstantially interact with homodimeric or non-IL-35R heterodimericcomplexes which comprise IL12Rβ2 or gp130 in a statistically significantamount. By “specifically or selectively binds to an IL-35R complex” isintended that the binding agent has a binding affinity for a non-IL-35Repitope which is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% ofthe binding affinity for the unique IL-35R epitope. One of skill will beaware of the proper controls that are needed to carry out such adetermination. An IL-35R specific binding agent may or may not modulatethe activity of IL-35R.

By “IL-35R specific binding/modulating agent” is intended an agent thatpossesses the properties of both an IL-35R specific binding agent and anIL-35R specific modulating agent. The IL-35R specific binding and/ormodulating agent can be an IL-35R agonist or an IL-35R antagonist.

By “IL-35 activation” is intended any activity resulting from thebinding of IL-35 to the IL-35R complex. As used herein, an agent that“specifically inhibits” IL-35 activity of the IL-35R complex willsubstantially block the activity of IL-35R by IL-35, but will notsignificantly block the activity of IL-35R by a non-IL-35 ligand.

In one embodiment, the IL-35R binding and/or modulating agent is a smallmolecule. For example, such small molecules include, but are not limitedto, peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heterorganicand organometallic compounds).

3. Anti-IL-35R Antibodies

As noted herein, the invention includes antibodies that specificallybind to the IL-35R complex. Antibodies, including monoclonal antibodies(mAbs), can be made by standard protocols. See, for example, Harlow andLane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.Briefly, a mammal such as a mouse, hamster or rabbit can be immunizedwith an immunogenic form of a peptide. Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques, well known in the art. In preferred embodiments,the subject antibodies are immunospecific for the unique antigenicdeterminants of IL-35R.

As discussed herein, these antibodies are collectively referred to as“anti-IL-35R antibodies”. Thus, by “anti-IL-35R antibodies” is intendedantibodies specific for IL-35R. All of these antibodies are encompassedby the discussion herein. The respective antibodies can be used alone orin combination in the methods of the invention.

By “antibodies that specifically bind” is intended that the antibodieswill not substantially cross react with another polypeptide. By “notsubstantially cross react” is intended that the antibody or fragment hasa binding affinity for a non-homologous protein which is less than 10%,less than 5%, or less than 1%, of the binding affinity for the IL-35Rcomplex.

In specific embodiments, the anti-IL-35R antibody binds specifically toIL-35R and further modulates the activity of the IL-35R complex. Thus,in specific embodiments, the anti-IL-35R antibody is an IL-35R agonistor is an IL-35R antagonist.

The anti-IL-35R antibodies disclosed herein and for use in the methodsof the present invention can be produced using any antibody productionmethod known to those of skill in the art. Thus, polyclonal sera may beprepared by conventional methods. In general, a solution containing theIL-35R complex or an active variant or fragment thereof is first used toimmunize a suitable animal, preferably a mouse, rat, rabbit, or goat.Rabbits or goats are preferred for the preparation of polyclonal seradue to the volume of serum obtainable, and the availability of labeledanti-rabbit and anti-goat antibodies.

Polyclonal sera can be prepared in a transgenic animal, preferably amouse bearing human immunoglobulin loci. In a preferred embodiment, Sf9(Spodoptera frugiperda) cells expressing IL-35R are used as theimmunogen Immunization can also be performed by mixing or emulsifyingthe antigen-containing solution in saline, preferably in an adjuvantsuch as Freund's complete adjuvant, and injecting the mixture oremulsion parenterally (generally subcutaneously or intramuscularly). Adose of 50-200 μg/injection is typically sufficient Immunization isgenerally boosted 2-6 weeks later with one or more injections of theprotein in saline, preferably using Freund's incomplete adjuvant. Onemay alternatively generate antibodies by in vitro immunization usingmethods known in the art, which for the purposes of this invention isconsidered equivalent to in vivo immunization. Polyclonal antisera areobtained by bleeding the immunized animal into a glass or plasticcontainer, incubating the blood at 25° C. for one hour, followed byincubating at 4° C. for 2-18 hours. The serum is recovered bycentrifugation (e.g., 1,000×g for 10 minutes). About 20-50 ml per bleedmay be obtained from rabbits.

Production of the Sf9 cells is disclosed in U.S. Pat. No. 6,004,552.Briefly, sequences encoding IL-35R complex are recombined into abaculovirus using transfer vectors. The plasmids are co-transfected withwild-type baculovirus DNA into Sf9 cells. Recombinantbaculovirus-infected Sf9 cells are identified and clonally purified.

Preferably the antibody is monoclonal in nature. By “monoclonalantibody” is an antibody obtained from a population of substantiallyhomogeneous antibodies, that is, the individual antibodies comprisingthe population are identical except for possible naturally occurringmutations that may be present in minor amounts. The term is not limitedregarding the species or source of the antibody. The term encompasseswhole immunoglobulins as well as fragments such as Fab, F(ab′)2, Fv, andothers which retain the antigen binding function of the antibody.Monoclonal antibodies are highly specific, being directed against asingle antigenic site on the target polypeptide. Furthermore, incontrast to conventional (polyclonal) antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. The modifier “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler and Milstein (Nature 256:495-97, 1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal. (Nature 352:624-28, 1991), Marks et al. (J. Mol. Biol. 222:581-97,1991) and U.S. Pat. No. 5,514,548.

By “epitope” is the part of an antigenic molecule to which an antibodyis produced and to which the antibody will bind. Epitopes can compriselinear amino acid residues (i.e., residues within the epitope arearranged sequentially one after another in a linear fashion), nonlinearamino acid residues (referred to herein as “nonlinear epitopes”—theseepitopes are not arranged sequentially), or both linear and nonlinearamino acid residues.

As discussed herein, mAbs can be prepared using the method of Kohler andMilstein, or a modification thereof. Typically, a mouse is immunizedwith a solution containing an antigen. Immunization can be performed bymixing or emulsifying the antigen-containing solution in saline,preferably in an adjuvant such as Freund's complete adjuvant, andinjecting the mixture or emulsion parenterally. Any method ofimmunization known in the art may be used to obtain the monoclonalantibodies of the invention. After immunization of the animal, thespleen (and optionally, several large lymph nodes) are removed anddissociated into single cells. The spleen cells may be screened byapplying a cell suspension to a plate or well coated with the antigen ofinterest. The B cells expressing membrane bound immunoglobulin specificfor the antigen bind to the plate and are not rinsed away. Resulting Bcells, or all dissociated spleen cells, are then induced to fuse withmyeloma cells to form hybridomas, and are cultured in a selectivemedium. The resulting cells are plated by serial dilution and areassayed for the production of antibodies that specifically bind theantigen of interest (and that do not bind to unrelated antigens). Theselected mAb-secreting hybridomas are then cultured either in vitro(e.g., in tissue culture bottles or hollow fiber reactors), or in vivo(as ascites in mice).

Where the anti-IL-35R antibodies of the invention are to be preparedusing recombinant DNA methods, the DNA encoding the monoclonalantibodies is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). The hybridoma cells described herein serve as apreferred source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding an antibody includes Skerra, A. (Curr. Opinion in Immunol.5:256-62, 1993) and Phickthun, A. (Immunol. Revs. 130:151-88, 1992).Alternatively, antibody can be produced in a cell line such as a CHOcell line, as disclosed in U.S. Pat. Nos. 5,545,403; 5,545,405 and5,998,144. Briefly the cell line is transfected with vectors capable ofexpressing a light chain and a heavy chain, respectively. Bytransfecting the two proteins on separate vectors, chimeric antibodiescan be produced. Another advantage is the correct glycosylation of theantibody.

Additionally, the term “anti-IL-35R antibody” as used herein encompasseschimeric and humanized anti-IL-35R antibodies. By “chimeric” antibodiesis intended antibodies that are most preferably derived usingrecombinant deoxyribonucleic acid techniques and which comprise bothhuman (including immunologically “related” species, e.g., chimpanzee)and non-human components. Thus, the constant region of the chimericantibody is most preferably substantially identical to the constantregion of a natural human antibody; the variable region of the chimericantibody is most preferably derived from a non-human source and has thedesired antigenic specificity to the IL-35R antigen. The non-humansource can be any vertebrate source that can be used to generateantibodies to a human IL-35R antigen or material comprising a humanIL-35R antigen. Such non-human sources include, but are not limited to,rodents (e.g., rabbit, rat, mouse, etc.; see, e.g., U.S. Pat. No.4,816,567) and non-human primates (e.g., Old World Monkeys, Apes, etc.;see, e.g., U.S. Pat. Nos. 5,750,105 and 5,756,096). As used herein, thephrase “immunologically active” when used in reference tochimeric/humanized anti-IL-35R antibodies means chimeric/humanizedantibodies that bind human IL-35R.

By “humanized” is intended forms of anti-IL-35R antibodies that containminimal sequence derived from non-human immunoglobulin sequences. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a hypervariable region (also known ascomplementarity determining region or CDR) of the recipient are replacedby residues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat, rabbit, or nonhuman primate having thedesired specificity, affinity, and capacity. The phrase “complementaritydetermining region” refers to amino acid sequences which together definethe binding affinity and specificity of the natural Fv region of anative immunoglobulin binding site. See, for example, Chothia et al. (J.Mol. Biol. 196:901-17, 1987) and Kabat et al. (U.S. Dept. of Health andHuman Services, NIH Publication No. 91-3242, 1991). The phrase “constantregion” refers to the portion of the antibody molecule that conferseffector functions.

Humanization can be essentially performed following the methodsdescribed by Jones et al. (Nature 321:522-25, 1986), Riechmann et al.(Nature 332:323-27, 1988) and Verhoeyen et al. (Science 239:1534-36,1988), by substituting rodent or mutant rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. See also U.S. Pat. Nos.5,225,539; 5,585,089; 5,693,761; 5,693,762; and 5,859,205. In someinstances, residues within the framework regions of one or more variableregions of the human immunoglobulin are replaced by correspondingnon-human residues (see, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762; and 6,180,370). Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance (e.g., to obtain desired affinity). In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.Accordingly, such “humanized” antibodies may include antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species.

Also encompassed by the term “anti-IL-35R antibodies” are xenogeneic ormodified anti-IL-35R antibodies produced in a non-human mammalian host,more particularly a transgenic mouse, characterized by inactivatedendogenous immunoglobulin loci. In such transgenic animals, competentendogenous genes for the expression of light and heavy subunits of hostimmunoglobulins are rendered non-functional and substituted with theanalogous human immunoglobulin loci. These transgenic animals producehuman antibodies in the substantial absence of light or heavy hostimmunoglobulin subunits. See, for example, U.S. Pat. Nos. 5,877,397 and5,939,598. Preferably, fully human antibodies to IL-35 can be obtainedby immunizing transgenic mice. One such mouse is disclosed in U.S. Pat.Nos. 6,075,181; 6,091,001; and 6,114,598.

Fragments of the anti-IL-35R antibodies are suitable for use in themethods of the invention so long as they retain the desired affinity ofthe full-length antibody. Thus, a fragment of an anti-IL-35R antibodywill retain the ability to specifically bind to IL-35R. Such fragmentsare characterized by properties similar to the corresponding full-lengthanti-IL-35R antibody; that is, the fragments will specifically bindIL-35R. Such fragments are referred to herein as “antigen-binding”fragments.

Suitable antigen-binding fragments of an antibody comprise a portion ofa full-length antibody, generally the antigen-binding or variable regionthereof. Examples of antibody fragments include, but are not limited to,Fab, F(ab′)₂, and Fv fragments and single-chain antibody molecules. By“Fab” is intended a monovalent antigen-binding fragment of animmunoglobulin that is composed of the light chain and part of the heavychain. By F(ab′)₂ is intended a bivalent antigen-binding fragment of animmunoglobulin that contains both light chains and part of both heavychains. By “single-chain Fv” or “sFv” antibody fragments is intendedfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. See, forexample, U.S. Pat. Nos. 4,946,778; 5,260,203; 5,455,030; and 5,856,456.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the sFv to form thedesired structure for antigen binding. For a review of sFv seePluckthun, A. (1994) in The Pharmacology of Monoclonal Antibodies, Vol.113, ed. Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in, for example,McCafferty et al. (Nature 348:552-54, 1990) and U.S. Pat. No. 5,514,548.Clackson et al. (Nature 352:624-28, 1991) and Marks et al. (J. Mol.Biol. 222:581-97, 1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology 10:779-83, 1992), as wellas combinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nucleic.Acids Res. 21:2265-66, 1993). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Methods 24:107-17, 1992 and Brennan et al., Science 229:81-3,1985). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-67, 1992). According to another approach, F(ab′)₂fragments can be isolated directly from recombinant host cell culture.Other techniques for the production of antibody fragments will beapparent to the skilled practitioner.

A representative assay to detect anti-IL-35R antibodies specific to theunique epitopes form upon complex formation of IL-35R is a “competitivebinding assay.” Competitive binding assays are serological assays inwhich unknowns are detected and quantitated by their ability to inhibitthe binding of a labeled known ligand to its specific antibody.Antibodies employed in such immunoassays may be labeled or unlabeled.Unlabeled antibodies may be employed in agglutination; labeledantibodies may be employed in a wide variety of assays, employing a widevariety of labels. Detection of the formation of an antibody-antigencomplex between an anti-IL-35R antibody and an epitope of interest canbe facilitated by attaching a detectable substance to the antibody.Suitable detection means include the use of labels such asradionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Such labeled reagents may be used in a variety of well-knownassays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA,fluorescent immunoassays, and the like. See, for example, U.S. Pat. Nos.3,766,162; 3,791,932; 3,817,837; and 4,233,402.

In still further embodiments, the antibody is bispecific, wherein afirst antigen binding domain specifically interacts with an epitope ofgp130 and said second antigen binding domain specifically interacts withan epitope of IL12Rβ2.

Further provided is a mixture of a first and a second antibody. Themixture comprise a first antibody having a first chemical moiety and thefirst antibody binds substantially only to gp130 and a second antibodyhaving a second chemical moiety and the second antibody bindsubstantially only to a second polypeptide comprising IL12Rβ2. The firstand the second chemical moiety allow for the interaction of said firstand said second antibody at an IL-35R complex to be detected. Methodsfor such forms of detection and chemical moieties of interest arediscussed elsewhere herein.

4. Anti-gp130 and Anti-IL12Rβ2Antibodies

The compositions further include antibodies that specifically bind tothe constituents of the IL-35R complex: gp130 and IL12Rβ2. As describedabove, techniques for conferring immunogenicity on a protein or peptideinclude conjugation to carriers or other techniques, well known in theart. In preferred embodiments, anti-gp130 and anti-IL12Rβ2 antibodiesare immunospecific for the unique antigenic determinants of gp130 andIL12Rβ2, respectively. The term “anti-gp130 antibody” as used hereinencompasses an antibody that binds substantially only to a gp130polypeptide or a biologically active variant or fragment thereof, wherethe antibody behaves as a specific modulating agent for IL-35R andsubstantially inhibits IL-35 activation of the IL-35R complex. The term“anti-IL12Rβ2_antibody” as used herein encompasses an antibody thatbinds substantially only to an IL12Rβ2 polypeptide or a biologicallyactive variant or fragment thereof, where such an antibody behaves as aspecific modulating agent for IL-35R and substantially inhibits IL-35activation of the IL-35R complex. In some embodiments, anti-gp130 andanti-IL12Rβ2_antibodies can be “specific modulating agents” whichmodulate the ability of the IL-35R to be activated by IL-35 but permitthe IL-35R complex to be activated by other non-IL-35 ligands. Thus, insome embodiments, an anti-gp130 antibody can bind substantially only toa gp130 polypeptide, or a biologically active variant or fragmentthereof, and act as a specific modulating agent for IL-35R bysubstantially and specifically inhibiting IL-35 activation of the IL-35Rcomplex. In such cases, IL-35R activation by non-IL-35 ligands caninclude, for example, the binding of Interleukin 27 (IL-27) orinterleukin 12 (IL-12) to the IL-35R complex. Methodologies providedherein for the production and use of anti-IL-35R antibodies can beadapted to make and use anti-gp130 and anti-IL12Rβ2 antibodies,

vi. Expression Cassettes and Host Cells

The various polynucleotides of the invention can be expressed in anexpression cassette. An expression cassette comprises one or moreregulatory sequences, selected on the basis of the cells to be used forexpression, operably linked to the desired polynucleotide. “Operablylinked” is intended to mean that the desired polynucleotide (i.e., gp130and/or IL12Rβ2 or active variants and fragments thereof) is linked tothe regulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a cell when the expression cassette or vector is introducedinto a cell). “Regulatory sequences” include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). See,for example, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells, those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences), or those that direct expressionof the polynucleotide in the presence of an appropriate inducer(inducible promoter). It will be appreciated by those skilled in the artthat the design of the expression cassette can depend on such factors asthe choice of the host cell to be transformed, the level of expressionof the polynucleotide that is desired, and the like. Such expressioncassettes typically include one or more appropriately positioned sitesfor restriction enzymes, to facilitate introduction of the nucleic acidinto a vector.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.Alternatively, a sequence that is heterologous to a cell is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified in the cell from its native form in compositionand/or genomic locus by deliberate human intervention.

It will further be appreciated that appropriate promoter and/orregulatory elements can readily be selected to allow expression of therelevant transcription units in the cell of interest. In certainembodiments, the promoter utilized to direct intracellular expression ofa silencing element is a promoter for RNA polymerase III (Pol III).References discussing various Pol III promoters, include, for example,Yu et al. (2002) Proc. Natl. Acad. Sci. 99(9), 6047-6052; Sui et al.(2002) Proc. Natl. Acad. Sci. 99(8), 5515-5520 (2002); Paddison et al.(2002) Genes and Dev. 16, 948-958; Brummelkamp et al. (2002) Science296, 550-553; Miyagashi (2002) Biotech. 20, 497-500; Paul et al. (2002)Nat. Biotech. 20, 505-508; Tuschl et al. (2002) Nat. Biotech. 20,446-448. According to other embodiments, a promoter for RNA polymeraseI, e.g., a tRNA promoter, can be used. See McCown et al. (2003) Virology313(2):514-24; Kawasaki (2003) Nucleic Acids Res. 31 (2):700-7.

The regulatory sequences can also be provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitableexpression systems for both prokaryotic and eukaryotic cells, seeChapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). See, Goeddel (1990) in Gene Expression Technology:Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

Various constitutive promoters are known. For example, in variousembodiments, the human cytomegalovirus (CMV) immediate early genepromoter, the SV40 early promoter, the Rous sarcoma virus long terminalrepeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase can be used to obtain high-level expression of the codingsequence of interest. The use of other viral or mammalian cellular orbacterial phage promoters which are well-known in the art to helpachieve expression of a coding sequence of interest. Promoters which maybe used include, but are not limited to, the long terminal repeat asdescribed in Squinto et al. (1991) Cell 65:1 20); the SV40 earlypromoter region (Bernoist and Chambon (1981) Nature 290:304 310), theCMV promoter, the M-MuLV 5′ terminal repeat the promoter contained inthe 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.(1980) Cell 22:787 797), the herpes thymidine kinase promoter (Wagner etal. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:144 1445), the regulatorysequences of the metallothionine gene (Brinster et al. (1982) Nature296:39 42); the following animal transcriptional control regions, whichexhibit tissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al. (1984) Cell 38:639 646; Ornitz et al. (1986) ColdSpring Harbor Symp. Quant. Biol. 50:399 409; MacDonald, 1987, HepatologyZ:425 515); insulin gene control region which is active in pancreaticbeta cells (Hanahan (1985) Nature 315:115 122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.(1984) Cell 38:647 658; Adames et al (1985) Nature 318:533 538;Alexander et al. (1987) Mol. Cell. Biol. 7:1436 1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al. (1986) Cell 45:485 495).

Inducible promoters are also known. Non-limiting examples of induciblepromoters and their inducer inlcude MT II/Phorbol Ester (TPA) (Palmiteret al. (1982) Nature 300:611) and heavy metals (Haslinger and Karin(1985) Proc. Nat'l Acad. Sci. USA. 82:8572; Searle et al. (1985) Mol.Cell. Biol. 5:1480; Stuart et al. (1985) Nature 317:828; Imagawa et al.(1987) Cell 51:251; Karin et al. (1987) Mol. Cell. Biol. 7:606; Angel etal. (1987) Cell 49:729; McNeall et al. (1989) Gene 76:8); MMTV (mousemammary tumor virus)/Glucocorticoids (Huang et al. (1981) Cell 27:245;Lee et al. (1981) Nature 294:228; Majors and Varmus (1983) Proc. Nat'lAcad. Sci. USA. 80:5866; Chandler et al. (1983) Cell 33:489; Ponta etal. (1985) Proc. Nat'l Acad. Sci. USA. 82:1020; Sakai et al. (1988)Genes and Dev. 2:1144); β-Interferon/poly(rI)X and poly(rc) (Tavernieret al. (1983) Nature 301:634); Adenovirus 5 E2/E1A (Imperiale and Nevins(1984) Mol. Cell. Biol. 4:875); c-jun/Phorbol Ester (TPA), H₂O₂;Collagenase/Phorbol Ester (TPA) (Angel et al. (1987) Mol. Cell. Biol.7:2256); Stromelysin/Phorbol Ester (TPA), IL-1 (Angel et al. (1987) Cell49:729); SV40/Phorbol Ester (TPA) (Angel et al. (1987) Cell 49:729);Murine MX Gene/Interferon, Newcastle Disease Virus; GRP78 Gene/A23187(Resendez Jr. et al. (1988) Mol. Cell. Biol. 8:4579);α-2-Macroglobulin/IL-6; Vimentin/Serum (Kunz et al. (1989) Nucl. AcidsRes. 17:1121); MHC Class I Gene H-2 kB/Interferon (Blanar et al. (1989)EMBO J. 8:1139); HSP70/E1a, SV40 Large T Antigen (Taylor and Kingston(1990) Mol. Cell. Biol. 10:165; Taylor and Kingston (1990) Mol. Cell.Biol. 10:176; Taylor et al. (1989) J. Biol. Chem. 264:15160);Proliferin/Phorbol Ester-TPA (Mordacq and Linzer (1989) Genes and Dev.3:760); Tumor Necrosis Factor/PMA (Hensel et al. (1989) Lymphokine Res.8:347); Thyroid Stimulating Hormone α Gene/Thyroid Hormone (Chatterjeeet al. (1989) Proc. Nat'l Acad. Sci. USA. 86:9114); and, Insulin EBox/Glucose.

Such expression cassettes can be contained in a vector which allow forthe introduction of the expression cassette into a cell. In specificembodiments, the vector allows for autonomous replication of theexpression cassette in a cell or may be integrated into the genome of acell. Such vectors are replicated along with the host genome (e.g.,nonepisomal mammalian vectors). In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors). However, the invention is intended to include such otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses, and adeno-associated viruses).See, for example, U.S. Publication 2005214851, herein incorporated byreference.

Any expression cassette can further comprise a selection marker. As usedherein, the term “selection marker” comprises any polynucleotide, whichwhen expressed in a cell allows for the selection of the transformedcell with the vector. For example, a selection marker can conferresistance to a drug, a nutritional requirement, or a cytotoxic drug. Aselection marker can also induce a selectable phenotype such asfluorescence or a color deposit. A “positive selection marker” allows acell expressing the marker to survive against a selective agent and thusconfers a positive selection characteristic onto the cell expressingthat marker. Positive selection marker/agents include, for example,Neo/G418, Neo/Kanamycin, Hyg/Hygromycin, hisD/Histidinol, Gpt/Xanthine,Ble/Bleomycin, HPRT/Hypoxanthine. Other positive selection markersinclude DNA sequences encoding membrane bound polypeptides. Suchpolypeptides are well known to those skilled in the art and cancomprise, for example, a secretory sequence, an extracellular domain, atransmembrane domain and an intracellular domain. When expressed as apositive selection marker, such polypeptides associate with the cellmembrane. Fluorescently labeled antibodies specific for theextracellular domain may then be used in a fluorescence activated cellsorter (FACS) to select for cells expressing the membrane boundpolypeptide. FACS selection may occur before or after negativeselection.

A “negative selection marker” allows the cell expressing the marker tonot survive against a selective agent and thus confers a negativeselection characteristic onto the cell expressing the marker. Negativeselection marker/agents include, for example, HSV-tk/Acyclovir orGancyclovir or FIAU, Hprt/6-thioguanine, Gpt/6-thioxanthine, cytosinedeaminase/5-fluoro-cytosine, diphtheria toxin or the ricin toxin. See,for example, U.S. Pat. No. 5,464,764, herein incorporated by reference.

In preparing an expression cassette or a homologous recombinationcassette, the various DNA fragments may be manipulated, so as to providefor the DNA sequences in the proper orientation and, as appropriate, inthe proper reading frame. Toward this end, adapters or linkers may beemployed to join the DNA fragments or other manipulations may beinvolved to provide for convenient restriction sites, removal ofsuperfluous DNA, removal of restriction sites, or the like. For thispurpose, in vitro mutagenesis, primer repair, restriction, annealing,resubstitutions, e.g., transitions and transversions, may be involved.

An “isolated” or “purified” polynucleotide or protein, or biologicallyactive portion thereof, is substantially or essentially free fromcomponents that normally accompany or interact with the polynucleotideor protein as found in its naturally occurring environment. Thus, anisolated or purified protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. An “isolated” polynucleotide is free ofsequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides, can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

Any cell can be used in the methods and compositions. In specificembodiments, the cell is from a mammal, a primate, a human, a domesticanimal or an agricultural animal. In specific embodiment, the cell is anon-human cell. Non-limiting animals that the cell can be derived frominclude cattle, sheep, goats, pigs, horses, rabbits, dogs, monkeys,cats, large felines (lions, tigers, etc.), wolves, mice, rats, rabbits,deer, mules, bears, cows, pigs, horses, oxen, zebras, elephants, and soon. The cell can further be from a plant, a worm (e.g., C. elegans), aninsect, a fish, a reptile, an amphibian, a bird (including, but notlimited to chickens, turkeys, ducks, geese and the like), a marsupial,etc. The cells can be derived from any tissue (diseased or healthy) fromany of these organisms. Expression of IL-35R can be engineered to occurin any cell type that one would want to control growth or proliferationof, especially tumor cells or tissues/cells that are common targets ofautoimmune diseases. Such host cells include cultured cells (in vitro),explants and primary cultures (in vitro and ex vivo).

The present invention further provides transgenic animals expressing afirst heterologous polynucleotide encoding gp130 or an active variant orfragment thereof and a second heterologous polynucleotide encoding anIL12Rβ2 polypeptide or an active variant or fragment thereof.

Such animals are useful as animal models having modulated IL-35Ractivity, including for example, animal models having modulated effectorT cell function. In general, methods of generating transgenic animalsand transformed cell lines are well known in the art (for example, seeGrosveld et al., Transgenic Animals, Academic Press Ltd., San Diego,Calif. (1992)). Using the nucleotide sequences disclosed herein encodinggp130 and IL12Rβ2, a skilled artisan can readily generate transgenicanimals and transformed cell lines which contain and express bothheterologous sequences. Such animals serve as models for the developmentof alternative therapies for therapies that modulate effector T cellfunction.

Such methods of the invention involve introducing a polypeptide orpolynucleotide into a cell. “Introducing” is intended to mean presentingto the cell the polynucleotide or polypeptide in such a manner that thesequence gains access to the interior of a cell of the plant. Themethods of the invention do not depend on a particular method forintroducing a sequence into a cell, only that the polynucleotide orpolypeptides gains access to the interior of a cell. Methods forintroducing polynucleotide or polypeptides into various cell types areknown in the art including, but not limited to, stable transformationmethods, transient transformation methods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a cell integrates into the DNA of the cell andis capable of being inherited by the progeny thereof “Transienttransformation” is intended to mean that a polynucleotide is introducedinto the cell and does not integrate into the genome of the cell or apolypeptide is introduced into a cell. Transformation protocols as wellas protocols for introducing polypeptides or polynucleotide sequencesinto cell may vary depending on the type of cell targeted fortransformation.

Exemplary art-recognized techniques for introducing foreignpolynucleotides into a host cell, include, but are not limited to,calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, particle gun, orelectroporation and viral vectors. Suitable methods for transforming ortransfecting host cells can be found in U.S. Pat. No. 5,049,386, U.S.Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355, Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and other standard molecular biologylaboratory manuals. Various transfection agents can be used in thesetechniques. Such agent are known, see for example, WO 2005012487. One ofskill will recognize that depending on the method by which apolynucleotide is introduced into a cell, the silencing element can bestably incorporated into the genome of the cell, replicated on anautonomous vector or plasmid, or present transiently in the cell.

Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of viral vector procedures, see Anderson (1992) Science256:808-813; Haddada et al. (1995) Current Topics in Microbiology andImmunology Doerfler and Bohm (eds); and Yu et al. (1994) Gene Therapy1:13-26. Conventional viral based systems for the delivery ofpolynucleotides could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.

II. Uses, Methods, and Kits

The polynucleotide(s) encoding the IL-35R complex and active variantsand fragments thereof, the IL-35R complex and active variants andfragments thereof, the soluble form of the IL-35R complex, the IL-35Rspecific binding and/or modulating agents, and the IL-35R agonist andantagonists disclosed herein can be used in one or more of the followingmethods: (a) screening assays; (b) detection assays; (c) predictivemedicine (e.g., diagnostic assays, prognostic assays, monitoringclinical trials, and pharmacogenomics); and (d) methods of treatment(e.g., therapeutic and prophylactic).

i. Methods to Screen for IL-35R Binding and/or Modulating

The invention provides a method (also referred to herein as a “screeningassay”) for identifying binding and/or modulating agents of IL-35R. Asdiscussed above, identification of various IL-35R binding agents are ofinterest including agonist IL-35R binding agents, antagonist IL-35Rbinding agents, and IL-35R specific binding agents. Similarly,identification of various IL-35R modulating agents are of interestincluding, for example, IL-35R agonist and antagonists.

The test compounds employed in the various screening assays can includeany candidate compound including, for example, peptides,peptidomimetics, small molecules, antibodies, or other drugs. Such testcompounds can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including biologicallibraries, spatially addressable parallel solid phase or solution phaselibraries, synthetic library methods requiring deconvolution, the“one-bead one-compound” library method, and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, nonpeptide oligomer, or small molecule librariesof compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

Determining the ability of the test compound to bind to the IL-35Rcomplex can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the IL-35R complex or a biologically active portion thereofcan be determined by detecting the labeled compound in a complex. Forexample, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,test compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

In one embodiment, an assay is a cell-free assay comprising contactingan IL-35R complex or biologically active fragment or variant thereofwith a test compound and determining the ability of the test compound tobind to the IL-35R complex or the biologically active variant orfragment thereof. Binding of the test compound to the IL-35R complex canbe determined either directly or indirectly. An indirect assay couldinclude assaying for a modulation in IL-35R activity. In a furtherembodiment, the test or candidate compound specifically binds to orselectively binds to the IL-35R complex.

In another embodiment, the assay includes contacting the IL-35R complexor biologically active variant or fragment thereof with a known compoundthat binds to the IL-35R complex (such as its ligand, IL-35) to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to preferentially bind toIL-35R complex or biologically active fragment or variant thereof ascompared to the known compound.

In another embodiment, an assay comprises contacting the IL-35R complexor biologically active portion thereof with a test compound anddetermining the ability of the test compound to modulate (e.g.,stimulate or inhibit, act as an agonist or antagonist) the activity ofthe IL-35R complex or biologically active portion thereof. Determiningthe ability of the test compound to modulate the activity of an IL-35Rcomplex can be accomplished, for example, by determining the ability ofthe IL-35R complex to bind to its ligand, IL-35, as described above, fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of an IL-35Rcomplex can be accomplished by determining the ability of the IL-35Rcomplex to further modulate intercellular downstream pathways modulatedby IL-35R. Such activities are discussed elsewhere herein.

In some assays, it may be desirable to immobilize either an IL-35Rcomplex or a biologically active fragment or variant thereof or the testcompound to facilitate separation of complexed from uncomplexed forms ofthe IL-35R complex, as well as to accommodate automation of the assay.In one embodiment, a fusion protein can be provided that adds a domainthat allows the IL-35R complex or active fragment or variant thereof orthe test agent to be bound to a matrix. For example,glutathione-S-transferase/IL-35R complex fusion proteins orglutathione-S-transferase/IL-35R complex fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtitre plates, which are then combined withthe test compound, and the mixture incubated under conditions conduciveto complex formation (e.g., at physiological conditions for salt andpH). Following incubation, the beads or microtitre plate wells arewashed to remove any unbound components and complex formation ismeasured either directly or indirectly, for example, as described above.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either the IL-35Rcomplex or active fragment thereof or the test compound can beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated IL-35R complexes or active fragments thereof or test agentscan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated96-well plates (Pierce Chemicals).

In yet another aspect of the invention, the IL-35R complex can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with the IL-35R complex or activefragments and variants thereof and, in some embodiments, modulate IL-35Rcomplex activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

ii. Methods for Detecting

Various methods and compositions for detecting and/or determining thelevel of expression of a polynucleotide encoding gp130 and IL12Rβ2 in asample are provided. A biological sample can comprise any sample inwhich one desires to determine the level of expression of apolynucleotide encoding gp130 and IL12Rβ2 or one desires to detect orquantitate the level of the IL-35R complex. The term “biological sample”is intended to include tissues, cells, and biological fluids isolatedfrom a subject, as well as tissues, cells, and fluids present within asubject. Detection of the expression of IL-35R in any cell type thatexpresses IL-35R can be performed, including expression levels in eitherdiseased verses healthy tissue. That is, the detection method of theinvention can be used to detect gp130 mRNA or genomic DNA, IL12Rβ2 mRNAor genomic DNA, or the IL-35R complex in a biological sample in vitro,as well as, in vivo. For example, in vitro techniques for detection ofinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of the IL-35R complex include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. In vitro techniques for detection of genomic DNAinclude Southern hybridizations. Furthermore, in vivo techniques fordetection of the IL-35R complex include introducing into a subject alabeled anti-IL-35R specific antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

a. Detecting Polynucleotides

In one embodiment, a method for detecting the level of expression of apolynucleotide encoding gp130 or active variants and fragments thereofand IL12Rβ2 or active variants and fragments thereof in a samplecomprises contacting the sample with a) a first and a second primercapable of specifically amplifying a first amplicon comprising apolynucleotide encoding a gp130 polypeptide or an active variant orfragment thereof; and, b) a third and a fourth primer capable ofspecifically amplifying a second amplicon comprising a polynucleotideencoding an IL12Rβ2 polypeptide or an active variant or fragmentthereof; wherein the encoded polypeptides form a biologically activeIL-35R complex. The first and the second amplicon is amplified and thendetected.

In other embodiments, a method for detecting the level of expression ofa polynucleotide encoding gp130 or active variants and fragments thereofand IL12Rβ2 or active variants and fragments thereof in a samplecomprises contacting the sample with a) a first polynucleotide capableof specifically detecting a polynucleotide encoding a gp130 polypeptideor an active variant or fragment thereof; and, b) a secondpolynucleotide capable of specifically detecting a polynucleotideencoding an IL12Rβ2 polypeptide or an active variant or fragmentthereof; wherein the encoded polypeptides form a biologically activeIL-35R complex; and detecting the polynucleotide encoding the gp130polypeptide or an active variant or fragment thereof and detecting thepolynucleotide encoding the IL12Rβ2 polypeptide or an active variant orfragment thereof.

In specific embodiments, the sample is contacted with a polynucleotideprobe that hybridizes under stringent hybridization conditions to thetarget sequences to be detected. The sample and probes are thensubjected the sample and probe to stringent hybridization conditions andthe hybridization of the probe to the target sequences is detected.

Primers and probes are based on the sequence of the polynucleotidesencoded by gp130 and IL12Rβ2 or active variants and fragments thereof.Any conventional nucleic acid hybridization or amplification method canbe used to identify the presence of the polynucleotides encoded by gp130and IL12Rβ2 in a sample. By “specifically detect” is intended that thepolynucleotide can be used either as a primer to specifically amplify anamplicon of a polynucleotide encoding gp130 or IL12Rβ2 or thepolynucleotide can be used as a probe that hybridizes under stringentconditions to a polynucleotide encoding gp130 or IL12Rβ2. The level ordegree of hybridization which allows for the specific detection or thespecific amplification of a polynucleotide encoding gp130 or IL12Rβ2 issufficient to distinguish the polynucleotide encoding gp130 or IL12Rβ2from a polynucleotide that does not encode the recited polypeptide. By“shares sufficient sequence identity or complementarity to allow for theamplification of a polynucleotide encoding gp130 or IL12Rβ2” is intendedthe sequence shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity or complementarity to a fragment oracross the full length of the polynucleotide encoding gp130 or IL12Rβ2.

Regarding the amplification of a target polynucleotide (e.g., by PCR)using a particular amplification primer pair, “stringent conditions” areconditions that permit the primer pair to hybridize to the targetpolynucleotide to which a primer having the corresponding wild-typesequence (or its complement) would bind and preferably to produce anidentifiable amplification product (the amplicon) in a DNA thermalamplification reaction. In a PCR approach, oligonucleotide primers canbe designed for use in PCR reactions to amplify a polynucleotideencoding gp130 or IL12Rβ2. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Methods of amplification arefurther described in U.S. Pat. Nos. 4,683,195, 4,683,202 and Chen et al.(1994) PNAS 91:5695-5699. These methods as well as other methods knownin the art of DNA amplification may be used in the practice of theembodiments of the present invention. It is understood that a number ofparameters in a specific PCR protocol may need to be adjusted tospecific laboratory conditions and may be slightly modified and yetallow for the collection of similar results. These adjustments will beapparent to a person skilled in the art.

The amplified polynucleotide (amplicon) can be of any length that allowsfor the detection of the polynucleotide encoding gp130 or IL12Rβ2. Forexample, the amplicon can be about 10, 50, 100, 200, 300, 500, 700, 100,2000, 3000, 4000, 5000 nucleotides in length or longer.

Any primer can be employed in the methods of the invention that allows apolynucleotide encoding gp130 or IL12Rβ2 to be amplified and/ordetected. For example, in specific embodiments, the first primer paircomprises primers comprising a fragment of a polynucleotide encodinggp130, wherein the first primer pair shares sufficient sequence identityor complementarity to the polynucleotide to amplify the polynucleotideencoding gp130; and, the second primer pair comprises primers comprisinga fragment of a polynucleotide encoding IL12Rβ2, wherein the firstprimer pair shares sufficient sequence identity or complementarity tothe polynucleotide to amplify the polynucleotide encoding IL12Rβ2. Inspecific embodiments, the primer can comprise at least 8, 10, 15, 20,25, 30, 40 or greater consecutive nucleotide of SEQ ID NO: 1, 2, 4 or 5.In order for a nucleic acid molecule to serve as a primer or probe itneed only be sufficiently complementary in sequence to be able to form astable double-stranded structure under the particular solvent and saltconcentrations employed.

In hybridization techniques, all or part of a polynucleotide thatselectively hybridizes to a target polynucleotide encoding gp130 orIL12Rβ2 is employed. By “stringent conditions” or “stringenthybridization conditions” when referring to a polynucleotide probe isintended conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of identity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length or lessthan 500 nucleotides in length.

As used herein, a substantially identical or complementary sequence is apolynucleotide that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6Xsodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Typically, stringent conditions for hybridization and detection will bethose in which the salt concentration is less than about 1.5 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringency conditions include hybridizationwith a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 40 to 45% formamide, 1.0M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl (1984)Anal. Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61(% form)-500/L; where M is the molarity of monovalent cations, % GC isthe percentage of guanosine and cytosine nucleotides in the DNA, % formis the percentage of formamide in the hybridization solution, and L isthe length of the hybrid in base pairs. The T_(m) is the temperature(under defined ionic strength and pH) at which 50% of a complementarytarget sequence hybridizes to a perfectly matched probe. T_(m) isreduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with >90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Haymes et al. (1985) In: NucleicAcid Hybridization, a Practical Approach, IRL Press, Washington, D.C.

A polynucleotide is said to be the “complement” of anotherpolynucleotide if they exhibit complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the polynucleotide molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions.

b. Detecting the IL-35R Complex

One aspect of the present invention relates to assays for detectingIL-35R complexes in the context of a biological sample. An exemplarymethod for detecting the presence or absence or the quantity of theIL-35R complex in a biological sample involves obtaining a biologicalsample and contacting the biological sample with a compound or an agentcapable of specifically binding and detecting an IL-35R complex, suchthat the presence of the IL-35R complex is detected in the biologicalsample. Results obtained with a biological sample from a test subjectmay be compared to results obtained with a biological sample from acontrol subject.

Detection of IL-35R with an IL-35R specific binding agent allows for thedetection, purification, and/or isolation of cell populations expressingIL-35R. Such methods find use in determining cell populations that aresensitive or resistant to the effects of IL-35. Information gained bysuch techniques can then be used when designing IL-35 treatments ortherapies.

In one embodiment, an agent for detecting the IL-35R complex is anantibody capable of specifically binding to the IL-35R complex,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(abN)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody.

iii. Kits

As used herein, “kit” refers to a set of reagents for the identificationand/or the detection of the polynucleotide encoding gp130 or IL12Rβ2 ordetection and/or quantitation of the IL-35R complex in biologicalsamples. The terms “kit” and “system,” as used herein are intended torefer to at least one or more detection reagents which, in specificembodiments, are in combination with one or more other types of elementsor components (e.g., other types of biochemical reagents, containers,packages, such as packaging intended for commercial sale, substrates towhich detection reagents are attached, electronic hardware components,instructions of use, and the like).

In one embodiment, a kit for determining the level of expression of apolynucleotide encoding gp130 and IL12Rβ2 in a sample is provided. Thekit comprises a) a first polynucleotide capable of specificallydetecting or amplifying a polynucleotide encoding a first polypeptideencoding gp130 or a biologically active variant or fragment thereof;and, b) a second polynucleotide capable of specifically detecting oramplifying a polynucleotide encoding IL12Rβ2 or a biologically activevariant or fragment thereof, wherein the encoded polypeptides form abiologically active IL-35R complex.

In specific embodiments, the kit comprises a) a first and a secondprimer that share sufficient sequence homology or complementarity to thepolynucleotide encoding gp130 or the active variant or fragment thereofto specifically amplify the polynucleotide encoding gp130; and, b) athird and a forth primer that share sufficient sequence homology orcomplementarity to a polynucleotide encoding IL12Rβ2 or an activevariant or fragment thereof to specifically amplify the polynucleotideencoding IL12Rβ2.

In still other embodiments, the kit comprises a) a first probe that canspecifically detect the polynucleotide encoding gp130 or the activevariant or fragment thereof, wherein the first probe comprises at leastone polynucleotide of a sufficient length of contiguous nucleotidesidentical or complementary to the polynucleotide encoding gp130 or theactive variant thereof; and, b) a second probe that can specificallydetect a second polynucleotide encoding IL12Rβ2 or an active variant orfragment thereof, wherein the second probe comprises at least onepolynucleotide of a sufficient length of contiguous nucleotidesidentical or complementary to a polynucleotide encoding IL12Rβ2 or anactive variant or fragment thereof. In still further embodiments, thefirst polynucleotide hybridizes under stringent conditions to thesequence encoding the gp130 polypeptide or active variant thereof; and,the second polynucleotide hybridizes under stringent conditions to thesequence encoding IL12Rβ2 or an active variant or fragment thereof.

In still other embodiments, a kit for determining the presence ofInterleukin 35 Receptor (IL-35R) in a sample is provided. Such a kit cancomprises any IL-35R specific binding and/or IL-35R specificbinding/modulating agent disclosed herein, including, but not limited toone or more of the IL-35R specific antibodies disclosed herein or anymixture thereof

iv. Methods for Modulating the Activity of the IL-35R Complex

Methods for modulating (i.e., inducing, inhibiting, potentiating,elevating, increasing, decreasing) the activity of the IL-35R complex ormodulating effector T-cell function are provided. Such methods cancomprise contacting a cell expressing the IL-35R complex with an IL-35Rantagonists or agonists.

As used herein, “responder T cells” or “effector T cells” refer to asubpopulation of mature T cells that facilitate an immune responsethrough cell activation and/or the secretion of cytokines. As usedherein, “effector T cells” include cytotoxic T cells (Tc), including forexample, CD8+ cells, and helper T cells (Th1 cells, Th2 cells, CD4+,Th17, Th9, and gamma delta T-cells). As used herein, “effector T cellfunction” includes an activity exerted by an effector T cell, asdetermined in vitro or in vivo, according to standard techniques. In oneembodiment, the effector T cell function includes the elimination of anantigen by, for example, the production of cytokines preferentiallyassociated with effector T cells, which modulate the activation of othercells, or by cytotoxic activity. In one embodiment, an effector T cellfunction is a cytotoxic (or cytolytic) T cell (Tc or CTL) function, suchas, for example, cytolysis of cells infected with microbes. In anotherembodiment, an effector T cell function is a Th1 cell function, e.g.,mediation of delayed type hypersensitivity responses and macrophageactivation. In yet another embodiment, an effector T cell function is aTh2 cell function, e.g., help to B cells (Mosmann et al. (1989) Annu.Rev. Immunol. 7, 145-173; Paul et al. (1994) Cell 76, 241-251; Arthurand Mason (1986) J Exp. Med. 163, 774-786; Paliard et al. (1988) J.Immunol. 141, 849-855; Finkelman et al., (1988) J. Immunol. 141,2335-2341). In another embodiment, an effector T cell function includesan inflammatory response, the suppression of immunological tolerance, or“tipping the balance” toward a proliferative/stimulatory environment.For purposes of the invention, effector T cell function is enhanced orinhibited by a statistically significant amount, for example, by atleast 5%, at least 10%, at least 15%, at least 20%, at least 30%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90% ascompared to an appropriate control cells.

An IL-35R agonist will act to suppress or inhibit effector T-cellactivity. The agonist can be, for example, an IL-35R specificbinding/modulating agent or an IL-35R specific modulating agent. By“inhibiting or suppressing an effector T cell function in a subject” isintended reducing and/or blocking of one or more of the functionsmediated by effector T cells. Thus, in one embodiment, a method ofsuppressing an effector T cell function is provided and comprisesadministering to the subject a therapeutically effective amount of anInterleukin 35 Receptor (IL-35R) agonist.

For example, an IL-35R agonist promotes immune tolerance, which can finduse, for example, in treating a subject having an autoimmune or aninflammatory disorder, including but not limited to, graft rejectionsand allergies. Thus, in one embodiment, a method of treating a subjecthaving an autoimmune or inflammatory disorder is provided. Such a methodcomprises administering to the subject a therapeutically effectiveamount of an agonist Interleukin 35 Receptor (IL-35R) agent. Variousagonist Interleukin 35 Receptor (IL-35R) agents and method for preparingsuch agents are discuses elsewhere herein. In specific embodiments, theagonist agent is an antibody or a small molecule. Examples of autoimmunediseases include, for example, type 1 diabetes, rheumatoid arthritis andmultiple sclerosis. Inflammatory disorders that may be treated include,for example, asthma and inflammatory bowel disease. In addition,limiting IL-35 by IL-35 antagonist could help enhance anti-tumorimmunity elicited by effector T cells.

In other embodiments, the IL-35R agonist can be used in combination witha therapeutic agent to reduce the immune response to the agent (i.e.,protein). For example, the IL-35R agonist can be used in combinationwith a therapeutic protein which must be chronically administered to asubject. Thus, in a specific embodiment, the method comprises includesadministering to the subject at least one additional therapeutic agentin combination with an IL-35R agonist. Such therapeutic agents, includebut are not limited to, a cytokine, a glucocorticoid, an anthracycline(e.g., doxorubicin or epirubicin), a fluoroquinolone (e.g.,ciprofloxacin), an antifolate (e.g., methotrexate), an antimetabolite(e.g., fluorouracil), a topoisomerase inhibitor (e.g., camptothecin,irinotecan or etoposide), an alkylating agent (e.g., cyclophosphamide,ifosfamide, mitolactol, or melphalan), an antiandrogen (e.g.,flutamide), an antiestrogen (e.g., tamoxifen), a platinum compound(e.g., cisplatin), a vinca alkaloid (e.g., vinorelbine, vinblastine orvindesine), a mitotic inhibitor (e.g., paclitaxel or docetaxel), aninhibitor of the PI3K/Akt/mTOR pathway, such as rapamycin, and/or aninhibitor of calcineurin.

An IL-35R antagonist will act to enhance or promote effector T-cellactivity. The antagonist can be, for example, an IL-35R specificbinding/modulating agent or an IL-35R specific modulating agent. By“enhancing an effector T cell function in a subject” is intendedreducing and/or blocking one or more of the functions mediated byeffector T cells. For example, an IL-35R antagonist will act to increaseor potentiate at least one effector T cell function and thereby increasethe immune response. Thus, in one embodiment, a method of increasing aneffector T cell function is provided and comprises administering to thesubject a therapeutically effective amount of an antagonisticInterleukin 35 Receptor (IL-35R) agent.

Such IL-35R antagonists find use in treating any conditions in which theIL-35 mediated activity of the T regulatory cells is shown to beblocking or limiting disease resolution. For example, the IL-35Rantagonists find use when activation of effector responses is desiredsuch as in cases of acute infection, vaccine response, anti-tumorimmunity or treating cancer. Thus, in one embodiment, a method oftreating a subject having a cancer or acute infection is provided. Sucha method comprises administering to the subject a therapeuticallyeffective amount of an antagonistic Interleukin 35 Receptor (IL-35R)agent. Various antagonistic Interleukin 35 Receptor (IL-35R) agents andmethod for preparing such agents are discuses elsewhere herein. Inspecific embodiments, the antagonist agent is an antibody or a smallmolecule.

It is further recognized that the various IL-35R antagonists can be usedin combination with an antigen to enhance the immune response to theantigen. For example, T effector cell responses employing an IL-35Rantagonist can be used to enhance a vaccine preparation. Thus, thevarious IL-35R antagonist are useful for increasing the efficacy ofanti-cancer vaccines or for vaccines that are poorly immunogenic.

Thus, further provided are methods for enhancing the efficacy orimmunogenicity of a vaccine in a subject, or overcoming a suppressedimmune response to a vaccine in a subject, including (i) administeringto the subject a therapeutically effective amount of an antagonistIL-35R agent and (ii) administering to the subject a vaccine. Inspecific embodiments, the antagonist is an IL-35R specificbinding/modulating agent. In one embodiment, the vaccine is a cancervaccine. For example, immune responses are suppressed in cancer andchronic infections and thus combining IL-35R agonists with therapeuticcancer vaccines or vaccines against chronic infections such as HCV, HIVand TB could improve efficacy.

By “vaccine” is intended a composition useful for stimulating a specificimmune response (or immunogenic response) in a subject. In someembodiments, the immunogenic response is protective or providesprotective immunity. For example, in the case of a disease-causingorganism the vaccine enables the subject to better resist infection withor disease progression from the organism against which the vaccine isdirected. Alternatively, in the case of a cancer, the vaccinestrengthens the subject's natural defenses against cancers that havealready developed. These types of vaccines may also prevent the furthergrowth of existing cancers, prevent the recurrence of treated cancers,and/or eliminate cancer cells not killed by prior treatments.

Representative vaccines include, but are not limited to, vaccinesagainst diphtheria, tetanus, pertussis, polio, measles, mumps, rubella,hepatitis B, Haemophilus influenzae type b, varicella, meningitis, humanimmunodeficiency virus, tuberculosis, Epstein Barr virus, malaria,hepatitis E, dengue, rotavirus, herpes, human papillomavirus, andcancers. Vaccines of interest include the two vaccines that have beenlicensed by the U.S. Food and Drug Administration to prevent virusinfections that can lead to cancer: the hepatitis B vaccine, whichprevents infection with the hepatitis B virus, an infectious agentassociated with liver cancer (MMWR Morb. Mortal. Wkly. Rep. 46:107-09,1997); and Gardasil™ which prevents infection with the two types ofhuman papillomavirus that together cause 70 percent of cervical cancercases worldwide (Speck and Tyring, Skin Therapy Lett. 11:1-3, 2006).Other treatment vaccines of interest include therapeutic vaccines forthe treatment of cervical cancer, follicular B cell non-Hodgkin'slymphoma, kidney cancer, cutaneous melanoma, ocular melanoma, prostatecancer, and multiple myeloma.

By “enhancing the efficacy” or “enhancing the immunogenicity” withregard to a vaccine is intended improving an outcome, for example, asmeasured by a change in a specific value, such as an increase or adecrease in a particular parameter of an activity of a vaccineassociated with protective immunity. In one embodiment, enhancementrefers to at least a 25%, 50%, 100% or greater than 100% increase in aparticular parameter. In another embodiment, enhancement refers to atleast a 25%, 50%, 100% or greater than 100% decrease in a particularparameter. In one example, enhancement of the efficacy/immunogenicity ofa vaccine refers to an increase in the ability of the vaccine to inhibitor treat disease progression, such as at least a 25%, 50%, 100%, orgreater than 100% increase in the effectiveness of the vaccine for thatpurpose. In a further example, enhancement of theefficacy/immunogenicity of a vaccine refers to an increase in theability of the vaccine to recruit the subject's natural defenses againstcancers that have already developed, such as at least a 25%, 50%, 100%,or greater than 100% increase in the effectiveness of the vaccine forthat purpose.

Similarly, by “overcoming a suppressed immune response” with regard to avaccine is intended improving an outcome, for example, as measured by achange in a specific value, such as a return to a formerly positivevalue in a particular parameter of an activity of a vaccine associatedwith protective immunity. In one embodiment, overcoming refers to atleast a 25%, 50%, 100% or greater than 100% increase in a particularparameter. In one example, overcoming a suppressed immune response to avaccine refers to a renewed ability of the vaccine to inhibit or treatdisease progression, such as at least a 25%, 50%, 100%, or greater than100% renewal in the effectiveness of the vaccine for that purpose. In afurther example, overcoming a suppressed immune response to a vaccinerefers to a renewed ability of the vaccine to recruit the subject'snatural defenses against cancers that have already developed, such as atleast a 25%, 50%, 100%, or greater than 100% renewal in theeffectiveness of the vaccine for that purpose.

A therapeutically effective amount of an IL-35R antagonist or agonistcan be administered to a subject. By “therapeutically effective amount”is intended an amount that is useful in the treatment, prevention ordiagnosis of a disease or condition. As used herein, a therapeuticallyeffective amount of an IL-R35 agonist or antagonist is an amount which,when administered to a subject, is sufficient to achieve a desiredeffect, such as modulating (inhibiting or promoting) effector T cellfunction in a subject being treated with that composition withoutcausing a substantial cytotoxic effect in the subject. The effectiveamount of an IL-35R— agonist or antagonist useful for modulatingeffector T-cell function will depend on the subject being treated, theseverity of the affliction, and the manner of administration of theIL-35R-agonist or antagonist.

By “subject” is intended mammals, e.g., primates, humans, agriculturaland domesticated animals such as, but not limited to, dogs, cats,cattle, horses, pigs, sheep, and the like. Preferably the subjectundergoing treatment with the pharmaceutical formulations of theinvention is a human.

When administration is for the purpose of treatment, administration maybe for either a prophylactic or therapeutic purpose. When providedprophylactically, the substance is provided in advance of any symptom.The prophylactic administration of the substance serves to prevent orattenuate any subsequent symptom. When provided therapeutically, thesubstance is provided at (or shortly after) the onset of a symptom. Thetherapeutic administration of the substance serves to attenuate anyactual symptom.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an IL-35R agonist or antagonist can include a singletreatment or, preferably, can include a series of treatments. It willalso be appreciated that the effective dosage of an IL-35R agonist orantagonist used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

It is understood that appropriate doses of such active compounds dependsupon a number of factors within the knowledge of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the activecompounds will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the active compound tohave upon the IL-35R complex. Exemplary doses include milligram ormicrogram amounts of the small molecule per kilogram of subject orsample weight (e.g., about 1 microgram per kilogram to about 500milligrams per kilogram, about 100 micrograms per kilogram to about 5milligrams per kilogram, or about 1 microgram per kilogram to about 50micrograms per kilogram. It is furthermore understood that appropriatedoses of an active agent depend upon the potency of the active agentwith respect to the expression or activity to be modulated. Suchappropriate doses may be determined using the assays described herein.When one or more of these small molecules is to be administered to ananimal (e.g., a human) in order to modulate activity of the IL-35Rcomplex, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

Therapeutically effective amounts of an IL-35R-specific binding and/ormodulating agent can be determined by animal studies. When animal assaysare used, a dosage is administered to provide a target tissueconcentration similar to that which has been shown to be effective inthe animal assays. It is recognized that the method of treatment maycomprise a single administration of a therapeutically effective amountor multiple administrations of a therapeutically effective amount of theIL-35R agonist or antagonist.

Any delivery system or treatment regimen that effectively achieves thedesired effect of modulating effector T cell function can be used. Thus,for example, formulations comprising an effective amount of apharmaceutical composition of the invention comprising IL-35R agonistsor antagonists can be used for the purpose of treatment, prevention, anddiagnosis of a number of clinical indications related to the activity ofthe IL-35R complex.

v. Pharmaceutical Compositions

The IL-35R complexes or active fragments and variants thereof, solubleforms of the IL-35R complex or active variants and fragments thereof,the IL-35R specific binding agents, and/or the IL-35R antagonist oragonists (also referred to herein as “active compounds”) disclosedherein can be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. In addition, it may bedesirable to administer a therapeutically effective amount of thepharmaceutical composition locally to an area in need of treatment(e.g., to an area of the body where inhibiting a regulatory T (T_(R))cell function is desired). This can be achieved by, for example, localor regional infusion or perfusion during surgery, topical application,injection, catheter, suppository, or implant (for example, implantsformed from porous, non-porous, or gelatinous materials, includingmembranes, such as sialastic membranes or fibers), and the like. In oneembodiment, administration can be by direct injection at the site (orformer site) of a cancer that is to be treated. In another embodiment,the therapeutically effective amount of the pharmaceutical compositionis delivered in a vesicle, such as liposomes (see, e.g., Langer, Science249:1527-33, 1990 and Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss,N.Y., pp. 353-65, 1989).

In yet another embodiment, the therapeutically effective amount of thepharmaceutical composition can be delivered in a controlled releasesystem. In one example, a pump can be used (see, e.g., Langer, Science249:1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14:201-40, 1987;Buchwald et al., Surgery 88:507-16, 1980; Saudek et al., N Engl. J. Med.321:574-79, 1989). In another example, polymeric materials can be used(see, e.g., Levy et al., Science 228:190-92, 1985; During et al., Ann.Neurol. 25:351-56, 1989; Howard et al., J. Neurosurg. 71:105-12, 1989).Other controlled release systems, such as those discussed by Langer(Science 249:1527-33, 1990), can also be used.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL∂ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

IV. Sequence Identity

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. It is further to be understood that all base sizesor amino acid sizes, and all molecular weight or molecular mass values,given for nucleic acids or polypeptides are approximate, and areprovided for description.

The subject matter of the present disclosure is further illustrated bythe following non-limiting examples.

EXPERIMENTAL Example 1 IL-35 Signaling and Suppression Mediated by IL-35Require the Expression of the IL-35R Materials and Methods:

Mice. C57BL/6 (wild type), CD4.cre, and IL12Rβ2^(−/−) mice werepurchased from the Jackson Laboratory. Gp130 floxed knockin mice wereprovided by Rodger McEver at Oklahoma Medical Research Foundation. gp130fl×CD4.cre×IL12Rb2^(−/−) were obtained by breeding the three mousestrains listed. All animal experiments were performed in AmericanAssociation for the Accreditation of Laboratory Animal Care-accredited,specific-pathogen-free facilities in the St. Jude Animal Resource Centerfollowing national, state and institutional guidelines. Animal protocolswere approved by the St Jude Animal Care and Use Committee.

T_(conv) Cell Purification. T_(eff)(CD4⁺CD25⁻CD45RB^(hi)) from thespleens and lymph nodes of C57BL/6 or knockout age-matchedgp130.fl×CD4.cre×IL12Rb2^(−/−) mice were positively sorted by FACS.After red blood cell lysis, cells were stained with antibodies againstCD4, CD25 and CD45RB and purified on a MoFlo cell sorter.

Transfection of HEK293T cells for IL-35 protein generation. IL-35constructs were generated by recombinant PCR and cloned into pPlGneo, apCIneo-based vector (Promega) that we have modified to include anIRES-GFP cassette. HEK293T cells were transfected using 10 μg plasmidper 2×10⁶ cells using Trans IT transfection reagent. Cells were sortedfor equivalent GFP expression and were cultured for 36 hours tofacilitate protein secretion. Dialyzed, filtered supernatant from cellswas used as the source of IL-35 in IL-35 mediated suppression assays.

iT_(R)35 conversion. iTr35 are an induced regulatory T cell populationthat is generated by treatment with IL-35 and suppress via IL-35. See,U.S. Provisional Application No. 61/156,995, herein incorporated byreference in its entirety. Purified murine T_(eff) cells were activatedby anti-CD3-+anti-CD28-coated latex beads in the presence of IL-35supernatant, at 25% of total culture medium, to induce “conversion” ofT_(eff) cells into iT_(R)35. Following conversion, iT_(R)35 werepurified for use in suppression assays.

In Vitro Suppression by IL-35 and iT_(R)35. T_(eff) were activated for72 hours with anti-CD3-+anti-CD28-coated latex beads in the presence ofIL-35 supernatant as 25%, 12.5%, or 6.25% of culture media. In parallel,iT_(R)35 were purified and assayed for their capacity to suppressfreshly sorted T_(eff) cell proliferation. Cultures were pulsed with 1mCi [³H]-thymidine for the final 8 hours of the 72 hour assay, and wereharvested with a Packard Micromate cell harvester. Counts per minutewere determined using a Packard Matrix 96 direct counter. Percentsuppression was calculated using the following formula: ((cpm of T_(eff)cells alone−cpm of T_(eff) cells treated with IL-35 or iT_(R)35)/cpm ofT_(eff) cells alone)*100.

Discussion/Conclusion:

FIG. 1 shows that wild-type T_(eff) proliferation is potently suppressedby IL-35 in a titratable manner. However, IL-35 is unable to suppressthe proliferation of T_(eff) cells that lack the IL-35R(gp130.fl×CD4.cre×IL12Rb2^(−/−)). iT_(R)35 suppression of T_(eff) cellproliferation is dependent upon IL-35. As such, IL-35R deficient T_(eff)cells are resistant to suppression mediated by iT_(R)35. These resultsdemonstrate that IL-35 signaling and suppression mediated by IL-35require the expression of the IL-35R.

Example 2 IL-35 Signals Primarily Through Two Different STAT Proteins,STAT1 and STAT4 Materials and Methods:

Mice. Spleens and lymph nodes from Il12rb1^(−/−) mice were provided byD. Fairweather and J. A. Frisancho (Johns Hopkins University),CD4^(cre)×gp130^(fl/fl) mice were provided by M. Karin and S.Grivennikov (University of California at San Diego), IL27ra^(−/−) micewere provided by C. Hunter and J. Stumhofer (University ofPennsylvania), Stat1^(−/−) mice were provided by A. Satoskar and P.Reville (Ohio State University), and Stat3^(−/−) mice were provided byC. Drake and H.R. Yen (Johns Hopkins University). IL12rb2^(−/−),Stat4^(−/−), Rag1^(−/−), C57BL/6, B6.PL and Balb/c mice were purchasedfrom the Jackson Laboratory. All animal experiments were performed inAmerican Association for the Accreditation of Laboratory AnimalCare-accredited, specific-pathogen-free facilities in the St. JudeAnimal Resource Center following national, state and institutionalguidelines. Animal protocols were approved by the St. Jude Animal Careand Use Committee.

Neutralizing IL-35 mAb. Neutralizing IL-35 mAb was developed byimmunization with recombinant murine Ebi3 protein. Briefly, recombinantmurine Ebi3 was cloned and expressed in a proprietary E. coli expressionsystem developed by Mike Jones (Shenandoah Biotechnology) and used forimmunization of Ebi3^(−/−) mice. Clones V1.4F5.29, V1.4H6.25, andV1.4C4.22 were subsequently chosen for their capacity to IP, blot, andspecifically neutralize IL-35 bioactivity.

Transfection of HEK293T cells for IL-35 and control protein generation.IL-35 constructs were generated by recombinant PCR and cloned intopPlGneo, a pCIneo-based vector (Promega) that has been modified toinclude an IRES-GFP cassette. A construct containing Ebi3 and 1112alinked by a flexible glycine-serine linker was used for IL-35 generationand an empty pPlGneo vector was used as a control. HEK293T cells weretransfected using 10 μg plasmid per 2×10⁶ cells using TransITtransfection reagent (Mirus). Transfection media was exchanged for freshculture media after 24 hours and were cultured for an additional 36hours to facilitate protein secretion. Dialyzed, filtered supernatantfrom cells was used at 25% of total culture medium to induce conversionof T_(conv) cells into iT_(R)35 or iT_(R)control cells.

Anti-CD3/CD28-coated latex beads. 4 μM sulfate latex beads (MolecularProbes) were incubated overnight at room temperature with rotation in a1:4 dilution of anti-CD3+ anti-CD28 antibody mix (13.3 μg/ml anti-CD3(murine clone #145-2c11, human clone #OKT3) (eBioscience) and 26.6 μg/mlanti-CD28 (murine clone #37.51, human clone #CD28.6) (eBioscience).Beads were washed 3 times with 5 mM phosphate buffer pH 6.5 andresuspended at 5×10⁷/ml in sterile phosphate buffer with 2 mM BSA.

Recombinant IL-35 beads. Beads were generated that presented IL-35 tocells in a manner that excluded use of 293T supernatants. Anti-p35 mAbclone 25806 (R&D Systems) or isotype control (rat IgG2) mAb was added to1 ml of IL-35 supernatant or control supernatant and rotated at 4° C.for 4 hours. Protein G beads were added and rotated for an additional12-18 hours. To ensure the protein was attached to the beads, the beadswere boiled to release bound protein, resolved by SDS-PAGE and probedwith anti-Ebi3 mAb. Both the beads and post IP supernatant were testedfor functional activity in a standard suppression assay. Beads werecultured with T_(conv) in medium containing anti-CD3+ anti-CD28conjugated beads as indicated for 3 days. Proliferation was determinedby [³H]-thymidine incorporation.

T_(conv) purification, iT_(R)35 conversion and suppressed T_(conv), cellgeneration. T_(conv) (CD4⁺CD25⁻CD45RB^(hi)) andT_(reg))(CD4⁺CD25⁺CD45RB^(lo) cells from the spleens and lymph nodes ofC57BL/6 or knockout age-matched mice were positively sorted by FACS.After red blood cell lysis, cells were stained with antibodies againstCD4, CD25 and CD45RB (Biolegend) and sorted on a MoFlo (Dako) orReflection (i-Cyt). Murine iT_(R)35 cells were generated. Briefly,purified murine T_(conv) cells from wild-type or indicated knockout micewere activated by anti-CD3-+anti-CD28-coated latex beads in the presenceof 25% culture medium from control or IL-35 transfected 293T cells(dialyzed against media and filtered) to generate murine iT_(R)35. Togenerate suppressed T_(conv), purified T_(conv) cells were activated inthe presence of anti-CD3-+anti-CD28-coated latex beads and T_(regs) at a4:1 (T_(conv):T_(reg) ratio) for 72 hours. Suppressed T_(conv) from theco-culture were re-sorted on the basis of congenic markers and used forqPCR analysis of receptor expression.

Immunoprecipitation and Western Blotting. Following 18 hour activationwith anti-CD3+anti-CD28 coated beads, cells were treated with 100 ng/mlIL12, IL27 or IL35 for indicated times. Whole cell lysates were lysed incold RIPA buffer and subjected to immunoblotting with antibodies forpSTAT1, pSTAT3, pSTAT4 and pSTATS (Cell Signaling Technology and SantaCruz Biotechnology). Blots were developed using ECL (AmershamBiosciences) and autoradiography.

In vitro proliferation and suppression assays. To determineproliferative capacity of cells generated as described above, 2.5×10⁴cells were activated with anti-CD3-+anti-CD28-coated latex beads for 72hours. Cultures were pulsed with 1 mCi [³H]-thymidine for the final 8hours of the 72 hour assay, and were harvested with a Packard Micromatecell harvester. Counts per minute were determined using a Packard Matrix96 direct counter (Packard Biosciences). For suppression assays, IL-35supernatants, IL-35 beads or iT_(R)35 were titrated into T_(conv) cellproliferation assays as indicated. Cultures were pulsed and harvested asdescribed for proliferation assays.

iT_(R)35-mediated control of homeostatic expansion. Homeostasis assayswere performed. Naive Thy1.2⁺ T_(conv) cells were isolated by FACS fromwild-type or knockout mice (as indicated) and used as “responder” cellsin adoptive transfer. Thy1.1⁺ iT_(R)35 were generated as described aboveand used as “suppressor” cells in adoptive transfer. T_(conv) cells(2×10⁶) with or without suppressor cells (5×10⁵) were resuspended in 0.5ml of PBS plus 2% FBS, and were injected intravenously through the tailvein into Rag1^(−/−) mice. Mice were euthanized seven days posttransfer, and splenocytes were counted, stained and analyzed by flowcytometry using antibodies against Thy1.1 and Thy1.2 (BD Bioscience).For each group, 5-10 mice were analyzed.

B16 tumor model. For T cell adoptive transfer experiments using the B16melanoma model, Rag1^(−/−) mice received indicated cells via the tailvein on day −1 of experiment. Wild type or receptor deficient naïveCD4⁺CD25⁻ (9×10⁶/mouse) and CD8⁺ T cells (6×10⁶/mouse) alone or incombination with iT_(R)35 cells (10⁶/mouse) were adoptively transferredinto mice. B16-F10 melanoma was a gift from Mary Jo Turk (DartmouthCollege, Hanover, N.H.) and was passaged intradermally (i.d.) in C57/B16mice 5 times to ensure reproducible growth. B16 cells were cultured inRPMI 1640 containing 7.5% FBS and washed three times with RPMI prior toinjections if viability exceeded 96%. Rag1^(−/−) mice were injected with120,000 cells on the right flank i.d. B16 tumor diameters were measureddaily with calipers and reported as mm³ (a²×b/2, where “a” is thesmaller caliper measurement and “b” the larger). For all experiments,B16 tumors were excised at day 14 when tumor size was 5-10 mm indiameter. For each group, 4-5 mice were analyzed.

The IL35 receptor comprises IL12Rβ2 and gp130.

To determine which IL12 family receptor chains are required for IL-35mediated suppression, three approaches were utilized, all of whichyielded similar results. The use of genetically deficient mice todetermine functions of proteins has been extremely useful in definingprotein activity. Therefore, it was first assessed whether IL-35 couldsuppress the proliferation of CD4⁺ T_(conv) cells that lacked expressionof each of the IL12 family receptor chains. T_(conv) cells purified byFACS from wild-type (C57BL/6), CD4^(crc)×gp130^(fl/fl) (abbreviatedgp130^(ΔT)), Il27ra^(−/−), Il12rb1^(−/−), Il12rb2^(−/−), orIl12rb2^(−/−)×CD4^(crc)×gp130^(fl/fl) (abbreviated IL35R^(ΔT)) mice wereactivated with anti-CD3-+anti-CD28-coated latex beads for 3 days in thepresence of indicated concentrations of IL-35 or iT_(R) ³⁵ incombination with neutralizing IL-35 mAb or isotype control mAb.Proliferation was determined by [³H]-thymidine incorporation. T_(conv)were treated with or without rIL-27 for 18 hours prior to analysis ofreceptor expression and proliferation. RNA was extracted, cDNA generatedand qPCR performed. Cytokine treated cells were mixed at indicatedconcentrations of IL-35 for 3 days. Proliferation was determined by[³H]-thymidine incorporation.

IL-35 can suppress the proliferation of both Il27ra^(−/−) andIl12rb1^(−/−) T_(covnv) cells to a degree similar to that seen inwild-type T_(conv) cells (data not shown). However, T_(conv) cells thatlack expression of either IL12Rβ2 (Il12rb2^(−/−)) or gp130(CD4^(crc)×gp130^(fl/fl); referred to herein as gp130^(ΔT)) arepartially resistant to IL-35 mediated suppression. Generation ofIl12rb2^(−/−) and CD4^(crc)×gp130″ mice (referred to herein asIL35R^(ΔT)) results in T_(conv) cells that are completely resistant toIL-35 mediated suppression. Many cytokines that signal through the gp130chain, including LIF, OncM and CNTF, require leukocyte inhibitoryfactor-β (LIFRβ) in addition to gp130 and the specificity-determiningreceptor chain. To determine whether IL-35 might also utilize LIFRβ,LIFRβ expression in T_(conv) cells was examined. Quantitative real-timePCR analysis suggests that T_(conv) cells, the targets of IL-35signaling, do not express LIFRβ, therefore it doesn't appear to beimportant for IL-35 signaling.

Second, IL-35 conjugated via an anti-p35 specific mAb, or isotypecontrol mAb, to Protein G beads was utilized as suppressors of T_(conv)cell proliferation. Isotype control or non-neutralizing anti-IL35 mAbwere incubated with IL-35 supernatant and then coupled with protein Gbeads. The protein G coupled beads were then incubated with T_(conv)cells activated in presence of a CD3 and a CD28. T_(conv) sorted fromindicated wild-type or receptor deficient T_(conv) cells were activatedin the presence of wild-type Tregs and proliferation determined by[³H]-thymidine incorporation. For a media alone control, nTreg, orT_(conv) cells mixed at a 4:1 ratio were activated in the presence ofanti-CD3-CD28-coated beads in the top chamber of a Transwell™ cultureplate. Responder T_(conv) were activated with anti-CD3-CD28-coated beadsin the bottom chamber of the plates. Proliferation of the responderT_(conv) cells in the bottom chambers was determined. No suppression ofproliferation was detected in isotype control beads, regardless ofgenotype. However, as seen with both IL-35 protein and iT_(R)35,suppression was limited in IL12Rβ2 and gp 130 deficient T_(conv) cellsand completely absent in T_(conv) cells that lack both IL12Rβ2 andgp130. It was previously shown that natural T_(regs) that lack IL-35expression (Ebi3^(−/−) or Il12a^(−/−)) are partially defective both invitro and in vivo (Collison et al. (2007) Nature 450:566-569).Therefore, it was expected that T_(conv) cells that lack the IL-35R and,thus, are unable to respond to IL-35, would be partially resistant toT_(reg)-mediated suppression. Indeed, gp130^(ΔT), Il12rb2^(−/−) andIL35R^(ΔT)T_(conv) cells are all partially resistant to T_(reg) mediatedsuppression of proliferation (data not shown). It was previously shownthat T_(conv) cells activated in the presence of T_(reg) are potentlysuppressive across a permeable membrane in an IL-35-dependent manner(Collison et al. (2009). J. Immunol. 182:6121-6128). Therefore, inaddition to a standard suppression assay, it was also determined whetherIL35R^(ΔT)T_(conv) were suppressed across a permeable membrane. Whilewild-type T_(conv) cells were potently suppressed by co-culturedT_(conv) and T_(reg), IL35R^(ΔT)T_(conv) cells were completely resistantto iT_(R) ³⁵ mediated suppression (data not shown).

Third, a novel induced T_(reg) population, iT_(R) ³⁵, has been describedthat suppresses the proliferation of T_(conv) cells exclusively viaIL-35 (Collison et al. (2010) Nature Immunology 11: 1093-1101). Bothexogenously added IL-35 and T_(reg) cells induce conversion of T_(conv)cells to iT_(R) ³⁵ in vitro, and in vivo, under inflammatory conditions.Given that their mode of suppression is by way of IL-35, they representa perfect tool for determining cell-mediated suppression via IL-35.Therefore, it was assessed whether iT_(R) ³⁵ suppressed theproliferation of each of the receptor deficient T_(conv) cells. WhileiT_(R) ³⁵ suppressed wild type, Il27ra^(−/−) and Il12rb1^(−/−) T_(conv)cells equally well, T_(conv) cells that lacked expression of eitherIL12Rβ2 or gp130 were partially resistant and cells that lacked bothIL12Rβ2 and gp130 were completely resistant to iT_(R)35 mediatedsuppression (data not shown). Moreover, neutralizing mAb to IL-35, butnot an isotype control, completely blocked the suppressive capacity ofiT_(R)35 of wild-type T_(conv) cells.

IL35R-Deficient T_(conv) Cells are Resistant to IL-35 MediatedSuppression In Vivo.

In the absence of their respective cytokine signaling receptor chains,in vivo cellular effects of IL-12 and IL-27 are completely abolished.Therefore, it was determined whether loss of the IL35R in vivo renders Tcells refractory to IL35-mediated suppression. Given that IL-35 iscentral to the suppression mediated by iT_(R)35, iT_(R)35 was utilizedin two different in vivo models to address this question. First,iT_(R)35 can control the homeostatic expansion of T_(conv) cells in thelymphopenic environment of the recombination activating gene-1(Rag1)^(−/−) mouse. Therefore, purified wild-type,gp130^(ΔT, Il)27ra^(−/−), Il12rb1^(−/−),Il12rb2^(−/−) orIL35R^(ΔT)Thy1.2⁺ T_(conv) cells, either alone or in the presence ofThy1.1⁺ iT_(R) ³⁵ cells were adoptively transferred into Rag1^(−/−)mice. Seven days post transfer, suppression of T_(conv) cell expansionwas monitored by determining the Thy1.2⁺ T_(conv) cell numbers. iT_(R)³⁵ cells significantly limited the proliferation of wild-type,gp130^(ΔT, Il)27ra^(−/−), Il12rb1^(−/−) and Il12rb2^(−/−)Thy1.2⁺T_(conv)cells. However, iT_(R) ³⁵ cells failed to block the expansion ofIL35R^(ΔT)Thy1.1^(+T) _(conv) cells (FIG. 2A). In the absence of onlyone receptor chain, in vivo biological activity of both IL-12 and IL-27is lost, it appears that IL-35 signaling in vivo is abrogated only byloss of both IL12Rβ2 and gp130 expression.

Second, it was previously shown that, like natural T_(regs), iT_(R) ³⁵can block the anti-tumor CD8⁺ T cell response against B16 melanoma.Wild-type, gp130^(ΔT), Il27ra^(−/−), Il12rb1^(−/−), Il12rb2^(−/−) orIL35R^(ΔT)CD4⁺ and CD8⁺ T cells, either alone or in the presence ofiT_(R) ³⁵ cells were adoptively transferred into Rag1^(−/−) mice. Thefollowing day, mice were inoculated intradermally with B 16 melanomacells and tumor size was monitored daily and reported after 14 days. Inthe absence of iT_(R) ³⁵, tumor burden was similar between micereceiving all CD4⁺ and CD8⁺ T cells, regardless of genotype (FIG. 2B).Tumor size was exacerbated in mice receiving iT_(R) ³⁵ cells incombination with wild-type, gp130^(ΔT), Il27ra^(−/−), Il12rb1^(−/−), andIl12rb2^(−/−)CD4⁺ and CD8⁺ T cells. However, IL35R^(ΔT) recipients werecompletely resistant to iT_(R) ³⁵ mediated prevention of tumor immunity.Collectively, these data clearly demonstrate that IL35R^(ΔT) cells areresistant to IL-35 mediated suppression in vivo.

IL-35 Signals Through STAT1 and STAT4

Given that IL12Rβ2 and gp130 constitute the IL-35 receptor, it washypothesized that the IL-35 signaling pathway might also overlap withthat of other cytokines that utilize these receptor chains. T_(conv)cells were activated in the presence of T_(reg) at a 4:1 ratio(responder:suppressor) for 72 hours. RNA was extracted and cDNAgenerated from resting or activated T_(conv) cells or from suppressedT_(conv) cells from T_(conv):T_(reg) co-cultures (resorted based ondifferential Thy1 markers). Relative gp130, Il27ra, Il12rb1 and Il12rb2mRNA expression was determined. Consistent with previous reports ref,IL-12 treatment of T_(conv) cells resulted in phosphorylation of STAT4and IL-27 signaling induced STAT1 and STAT3 phosphorylation (data notshown). Interestingly, wild-type T_(conv) cells, which are responsive toIL-35 mediated suppression, demonstrated phosphorylation of both STAT1and STAT4, but no activation of either STAT3 or STAT5. Moreover, noinduction of p-STAT1 or p-STAT4 was seen in T_(conv) cells that lack theIL-35 receptor (Il12rb2^(−/−)×gp130^(ΔT) T_(conv), hence forth referredto as IL35R^(ΔT)) (data not shown). To better determine the kinetics ofSTAT phosphorylation in response to IL-35 treatment, T_(conv) cells wereactivated for 24 hours with anti-CD3+anti-CD28 coated beads and treatedwith IL-35 for indicated times. Western blot analysis demonstrated thatp-STAT1 was the most dramatic, with maximal phosphorylation evident at30 minutes. Similar to STAT3 and STAT5, STAT4 phosphorylation was lesspronounced, but was sustained over the course of time analyzed. Todetermine which STATs were most critical to IL-35 signaling, T_(conv)cells that lack STAT1, STAT3, or STAT4 were utilized. Whereas IL-35 cansuppress the proliferation of Stat3^(−/−) T_(conv) cells to a degreesimilar to that seen in wild-type T_(conv) cells, suppression ofStat1^(−/−) and Stat4^(−/−) T_(conv), cells was reduced (data notshown). Similarly, T_(reg)-mediated suppression of both Stat1^(−/−) andStat4^(−/−) T_(conv) cells was impaired (data not shown). Reducedsignaling in Stat1^(−/−) and Stat4^(−/−) T_(conv) cells is not due tolack of receptor expression as mRNA expression of receptor chains issimilar in Stat1^(−/−), Stat4^(−/−) and wild-type T_(conv) cells (datanot shown). Collectively, these data suggest that STAT1 and STAT4 arecritical for IL-35 mediated signal transduction.

IL-35 is a Target of the IL-35 Signaling Pathway.

It has been previously shown that IL-35 can convert proliferative, IL35T_(conv) cells into hypo-responsive, strongly suppressive iT_(R)35 whichexpress and mediate suppression via IL-35 (Collison et al. (2010) NatureImmunology 11: 1093-1101). Activation of wild-type T_(conv) in thepresence of IL-35 significantly upregulated Ebi3 and 1112a mRNA, the twocomponents of IL-35 (Ebi3 and p35, respectively). Interestingly,gp130^(ΔT), Il12rb2^(−/−) and IL35R^(ΔT T) _(conv) cells are allresistant to induction of Ebi3 expression (FIG. 3A). However, gp130^(ΔT)T_(conv) cells retain the ability to upregulate 1112a expression inresponse to IL-35 treatment, suggesting that p35 expression may bedownstream of IL12Rβ2 signaling. Induction of IL-35 expression inresponse to IL-35 treatment is critical for conversion of T_(conv) cellsinto iT_(R) ³⁵. Therefore, the ability of receptor deficient mice to beconverted into iT_(R) ³⁵ was assessed. To determine whether IL-35treated T_(conv) cells had acquired regulatory capacity, they wereco-cultured as suppressors with freshly purified responder T_(conv)cells (FIG. 3B). T_(conv) cells treated with control protein, regardlessof genotype, were incapable of suppressing responder T_(conv) cellproliferation. Furthermore, wild-type, but not gp130^(ΔT), Il12rb2^(−/−)or IL35R^(ΔT)T_(conv9), cells were capable of suppressing T_(conv) cellproliferation. In addition, both Stat1^(−/−), Stat4^(−/−) T_(conv) cellsfail to upregulate expression of Ebi3 and 1112a to the same degree aswild-type T_(conv) cells (data not shown). Moreover, early induction ofEbi3 and 1112a mRNA expression, which peak at 3 hours and 1 hour,respectively, suggest that IL-35 is a direct target of IL-35 signaling(data not shown). Together, these results suggest that cells that lackthe IL-35 receptor or signaling components are unable to induce IL-35expression.

Discussion

Important similarities and interesting differences between IL-12, IL-27and IL-35 signaling have been illuminated by this study. Notsurprisingly, the IL-35 receptor and signaling pathway overlap with thatof IL-12 and IL-27. However, unlike its siblings, IL-35 appears to beable to signal, in part, through each of the receptor chains, IL-12Rβ2and gp130. This is likely due to the fact that each of these chains isthe signal transducing subunit of their respective cytokine receptors.In addition, like IL-27, IL-35 signals primarily through two differentSTAT proteins, STAT1 and STAT4. However, STAT3 appears dispensable forIL-35 signaling, an interesting observation given its importance forIL-27 signaling, which is downstream of gp130 engagement. In addition,STAT1/STAT3 heterodimers have been previously described yet there is noprecedent for STAT1/STAT 4 heterodimerization.

The expression pattern of the IL-35 receptor also provides insight intopotential IL-35 target cell types. While gp130 is fairly ubiquitouslyexpressed, both IL-12R chains are expressed mainly by activated T cellsand NK cells. In T cells, the expression of IL-12Rβ2 is confined to Th1cells, and its expression correlates with responsiveness to IL-12 andpresumably IL-35. Expression of IL-12Rβ2 has also been shown on othercell types, such as dendritic cells which would vastly affect the scopeof IL-35 bioactivity in the immune system. IL-12Rβ2 is undetectable onmost resting T cells, but can be rapidly upregulated by exposure toIL-12, IL-27, IFN-γ, tumor-necrosis factor (TNF) and co-stimulationthrough CD28. Thus, IL35 might have biological effects on a variety ofcellular targets and under a variety of disease conditions.

Since IL-35 appears to utilize receptor chains and STATs that aresimilar to those used by other IL-12 family members, another importantquestion is how a T cell can translate potentially similar signals intosuch distinct biological outcomes. Given the opposing activities ofIL-35 and IL-12, IL-23, and IL-27, it is possible that differentkinetics, binding affinities, or potentially as yet unidentifiedheterodimerization patterns may differentiate the signaling pathways insuch a way to mediate such diverse biological consequences.

TABLE 1 Summary of SEQ ID NOS SEQ ID NO Description Type of sequence 1gp130 Full length cDNA 2 gp130 DNA coding region 3 gp130 Amino acid 4IL12Rβ2 Full length cDNA 5 IL12Rβ2 DNA coding region 6 IL12Rβ2 Aminoacid

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A soluble Interleukin 35 receptor (IL-35R) complex comprising: a) a first polypeptide comprising the extracellular domain of a gp130 polypeptide or a biologically active variant or fragment thereof; and, b) a second polypeptide comprising the extracellular domain of an IL12Rβ2 polypeptide or a biologically active fragment or variant thereof; wherein said soluble complex bind IL-35.
 2. The soluble Interleukin 35 receptor (IL-35R) complex of claim 1, wherein a) said first polypeptide comprises the extracellular domain of SEQ ID NO:3, an active fragment thereof, or a sequence having at least 80% sequence identity to the extracellular domain of SEQ ID NO:3; and, b) said second polypeptide comprises the extracellular domain of SEQ ID NO:6 or an active fragment thereof or a sequence having at least 80% sequence identity to SEQ ID NO:6.
 3. A polynucleotide comprising a nucleotide sequence encoding a soluble Interleukin 35 receptor (IL-35R) complex comprising: a) a first polynucleotide encoding a first polypeptide comprising the extracellular domain a gp130 polypeptide or a biologically active fragment or variant thereof; and, b) a second polynucleotide encoding a second polypeptide comprising the extracellular domain of an IL12Rβ2 polypeptide or a biologically active variant or fragment thereof.
 4. The polynucleotide of claim 3, wherein, a) the first polynucleotide encoding the first polypeptide comprises the extracellular domain of SEQ ID NO:3, an active fragment thereof, or a sequence having at least 80% sequence identity to the extracellular domain of SEQ ID NO:3; and, b) the second polynucleotide encoding the second polypeptide comprises the extracellular domain of SEQ ID NO:6, an active fragment thereof, or a sequence having at least 80% sequence identity to SEQ ID NO:6.
 5. A mixture of polynucleotides encoding a soluble Interleukin 35 receptor (IL-35R) complex comprising: a) a first nucleotide sequence encoding a first polypeptide comprising the extracellular domain of a gp130 polypeptide or biologically active variant or fragment thereof; and, b) a second nucleotide sequence encoding a second polypeptide comprising the extracellular domain of an IL12Rβ2 polypeptide or a biologically active variant or fragment thereof; wherein said first and said second polypeptide form the soluble IL-35R complex that bind IL-35.
 6. The mixture of polynucleotides of claim 5, wherein a) the first nucleotide sequence encoding the first polypeptide comprises the extracellular domain of SEQ ID NO:3, an active fragment thereof or a sequence having at least 80% sequence identity to the extracellular domain of SEQ ID NO:3; and, b) the second nucleotide sequence encoding the second polypeptide comprises the extracellular domain of SEQ ID NO:6, an active fragment thereof, or a sequence having at least 80% sequence identity to SEQ ID NO:6.
 7. A non-human cell comprising the polynucleotide of claim
 3. 8. A transgenic animal having stably integrated into its genome the polynucleotide of claim
 3. 9. An isolated cell comprising the polynucleotide of claim
 3. 10. A pharmaceutical composition comprising the polypeptide of claim
 1. 11. An isolated Interleukin 35 receptor (IL-35R) complex comprising a) a first polypeptide comprising a gp130 polypeptide or a biologically active variant or fragment thereof; and, b) a second polypeptide comprising an Il12Rβ2 polypeptide or a biologically active variant or fragment thereof; wherein said complex has IL-35R activity.
 12. The isolated Interleukin 35 receptor (IL-35R) complex of claim 11, wherein a) the first polypeptide comprises SEQ ID NO:3 or a sequence having at least 80% sequence identity to the extracellular domain of SEQ ID NO:3; and, b) the second polypeptide comprises SEQ ID NO:6 or a sequence having at least 80% sequence identity to SEQ ID NO:6.
 13. An antibody that binds substantially only to the IL-35R complex of claim
 12. 14. (canceled)
 15. (canceled)
 16. The antibody of claim 13, wherein (a) said antibody is a monoclonal antibody; or, (b) said antibody is bispecific, wherein a first antigen binding domain specifically interacts with said first polypeptide and said second antigen binding domain specifically interacts with said second polypeptide.
 17. (canceled)
 18. An antibody that binds substantially only to the first polypeptide or a biologically active variant or fragment thereof of the IL-35R complex of claim 11, wherein (a) said antibody behaves as a specific modulating agent for IL-35R and substantially inhibits IL-35 activation of the IL-35R complex; or, (b) wherein said antibody behaves as a specific modulating agent for IL-35R and substantially inhibits IL-35 activation of the IL-35R complex.
 19. (canceled)
 20. A mixture of a first and a second antibody comprising: a) a first antibody having a first chemical moiety, said first antibody binds substantially only to a first polypeptide comprising a gp130 polypeptide or an active variant or fragment thereof; and, b) a second antibody having a second chemical moiety, said second antibody binds substantially only to a second polypeptide comprising a IL12Rβ2 polypeptide or a biologically active variant or fragment thereat c) wherein said first and said second chemical moiety allow for the interaction of said first and said second antibody at an IL-35R complex to be detected.
 21. The mixture of the first and the second antibody of claim 20, wherein a) said first antibody binds substantially only to the first polypeptide comprising SEQ ID NO:3 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3; and, b) said second antibody bind substantially only to a second polypeptide comprising SEQ ID NO:6 or the sequence having at least 80% sequence identity to SEQ ID NO:6.
 22. (canceled)
 23. (canceled)
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 27. (canceled)
 28. A kit for determining the level of expression of a polynucleotide encoding gp130 and a polynucleotide encoding IL12Rβ2 or a biologically active variant or fragment thereof in a sample comprising a) a first polynucleotide capable of specifically detecting or specifically amplifying a polynucleotide encoding a gp130 polypeptide or a biologically active variant or fragment thereof; and, h) a second polynucleotide capable of specifically detecting or specifically amplifying a polynucleotide encoding a IL12Rβ2 polypeptide or a biologically active variant or fragment thereof; wherein said encoded polypeptides form a biologically active IL-35R complex.
 29. The kit of claim 28, wherein a) the first polynucleotide is capable of specifically detecting or amplifying a polynucleotide encoding the amino acid sequence of SEQ ID NO:3 or a sequence having at least 80% sequence identity to SEQ ID NO:3; and, b) the second polynucleotide is capable of specifically detecting or amplifying a polynucleotide encoding the amino acid sequence of SEQ ID NO:6 or a sequence having at least 80% sequence identity to SEQ ID NO:6.
 30. The kit of claim 28, wherein said kit comprises a) a first and a second primer that share sufficient sequence homology or complementarity to said first polynucleotide to specifically amplify said first polynucleotide; and, b) a third and a forth primer that share sufficient sequence homology or complementarity to said second polynucleotide to specifically amplify said second polynucleotide.
 31. The kit of claim 28, wherein said kit comprises a) a first polynucleotide that can specifically detect said first polynucleotide, wherein said first polynucleotide comprises at least one DNA molecule of a sufficient length of contiguous nucleotides identical or complementary to SEQ ID NO:3; and, b) a second polynucleotide that can specifically detect said second polynucleotide, wherein said second polynucleotide comprises at least one DNA molecule of a sufficient length of contiguous nucleotides identical or complementary to SEQ ID NO:6.
 32. The kit of claim 28, wherein said kit comprises a) a first polynucleotide that hybridizes under stringent conditions to the sequence of SEQ ID NO:3; and, b) a second polynucleotide that hybridizes under stringent conditions to the sequence of SEQ ID NO:6.
 33. A kit for determining the presence of Interleukin 35 Receptor (IL-35R) in a sample comprising an antibody of claim
 13. 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. A method for detecting an IL-35R complex comprising a) contacting a sample with a compound which selectively binds to a IL-35R complex; and b) detecting a complex comprising the IL-35R complex and the compound; and thereby detecting said IL-35R complex.
 38. The method of claim 37, wherein the compound which binds to the polypeptide is an antibody.
 39. A method for (a) modulating the activity of an Interleukin 35 receptor (IL-35R) complex or increasing an immune response, or increasing the activity of an effector T cell function in a subject, comprising administering to the subject a therapeutically effective amount of a soluble IL-35R complex of claim 1; or, (b) treating a subject having a cancer or a chronic inflammatory disease, comprising administering to the subject a therapeutically effective amount of a soluble Interleukin 35 receptor (IL-35R) complex of claim 1; or, (c) increasing the activity of an effector T cell function in a subject, comprising administering to the subject a therapeutically effective amount of a soluble IL-35R complex of claim
 1. 40. (canceled)
 41. (canceled)
 42. A method to identify an Interleukin 35 receptor (IL-35R) binding agent comprising the steps of: a) contacting the IL-35R complex, or a cell expressing the IL-35R complex with a candidate compound; and b) determining whether the IL-35R complex binds to the candidate compound.
 43. The method of claim 42, wherein the binding of the candidate compound to the IL-35R complex is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of candidate compound/IL-35R binding; or b) detection of binding using a competition binding assay.
 44. (canceled)
 45. (canceled)
 46. (canceled)
 47. (canceled)
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 49. (canceled)
 50. A method for (a) screening for an Interleukin-35R (IL-35R) modulating agent comprising contacting IL-35R with a candidate compound and determining the effect of the test compound on the activity of the IL-3 5R complex to thereby identify a compound which modulates the activity of the IL-35R complex; or, (b) modulating the activity of an Interleukin 35 Receptor (IL-35R) comprising contacting a cell expressing said IL-35R with an IL-35R specific binding/modulating agent; or, (c) modulating the activity of an effector T cell function in a subject, comprising administering to the subject a therapeutically effective amount of an Interleukin 35 Receptor (IL-35R) specific binding/modulating agent or, (d) treating a subject having a cancer or a chronic inflammatory disease, comprising administering to the subject a therapeutically effective amount of an antagonistic Interleukin 35 Receptor (IL-35R) specific binding/modulating agent or, (e) treating a subject having an autoimmune or inflammatory disorder comprising administering to the subject a therapeutically effective amount of an agonist Interleukin 35 Receptor (IL-35R) specific binding/modulating agent.
 51. (canceled)
 52. (canceled)
 53. (canceled)
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 55. (canceled)
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 59. (canceled) 